Alleles of the human mu opioid receptor and diagnostic methods based thereon

ABSTRACT

Provided herein are variant alleles of a gene encoding a mu opioid receptor, along with cloning vectors for replicating such variant alleles, expressing vectors for expressing the variant alleles to produce variant mu opioid receptors, and antibodies to such variant receptors. Also disclosed are binding characteristics of such variant receptors regarding binding to opioid ligands, and the using of such binding characteristics to diagnose a subjects susceptibility to pain, susceptibility to an addictive disease, selecting an appropriate pain reliever along with a therapeutically effective amount of the reliever to administer to a subject suffering from pain. In addition, diagnostic methods for diagnosing a disease or disorder such as infertility, constipation, diarrhea, decreased immune response relative to a standard, and decreased ability to withstand stress relative to a standard, along with commercial kits for diagnosing such diseases or disorders. Furthermore, the invention is also directed to identification of targeted prevention methods, early therapeutic intervention, and improved treatment of opioid addiction, infertility, constipation, diarrhea, impaired immune responsiveness, and stress.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Provisional Application Ser. No.60/092,402, filed Jul. 10, 1998.

GOVERNMENTAL SUPPORT

This invention was made government support under Grant Nos. NIDARO1-DA09444, NIDA P50-DA05130, and NIDA K05-DA00049 awarded by theNational Institute of Drug Addiction. The Government has certain rightsin the invention.

FIELD OF THE INVENTION

This invention relates generally to alleles of the human mu opioidreceptor gene, along with products derived from such alleles. Alsoincluded herein are methods of diagnosing various susceptibilities usingsuch alleles and determining treatment for certain diseases based uponthe presence of specific alleles of the human mu opioid receptor gene,and various diseases or disorders related to physiological functionsregulated by the hypothalamus pituitary adrenal axis (HPA) or thehypothalamus pituitary gonadal axis (HPG).

BACKGROUND OF THE INVENTION

Opioid drugs have various effects on perception of pain, consciousness,motor control, mood, autonomic function, and can also induce physicaldependence. The endogenous opioid system plays an important role inmodulating endocrine, cardiovascular, respiratory, gastrointestinalfunctions, and immune functions. Opioids, either exogenous orendogenous, exert their actions by binding to specificmembrane-associated receptors.

Examples of exogenous opioids presently known include, opium, heroin,morphine, codeine, fentanyl, and methadone, to name only a few.Moreover, a family of over 20 endogenous opioid peptides has beenidentified, wherein the members possess common structural features,including a positive charge juxtaposed with an aromatic ring that isrequired for interaction with an opioid receptor. It has been determinedthat most, if not all the endogenous opioid peptides are derived fromthe proteolytic processing of three precursor proteins, i.e.,prbopiomelanocortin, proenkephalin, and prodynorphin. In addition, afourth class of endogenous opioids, the endorphins, has been identified(the gene encoding these proteins has not yet been cloned). In theprocessing of the endogenous opioid precursor proteins, initialcleavages are made by membrane-bound proteases that cut next to pairs ofpositively charged amino acid residues, and then trimming reactionsproduce the final endogenous opioids secreted from cells in vivo.Different cell types contain different processing enzymes so that, forexample proopiomelanocortin can be processed into different endogenouspeptides by different cells. For example, in the anterior lobe of thepituitary gland, only corticotropin (ACTH), β-lipotropin, andβ-endorphin are produced. Both pro-enkephalin and pro-dynorphin aresimilarly processed by specific enzymes in specific cells to yieldmultiple opioid peptides.

Pharnacological studies have suggested there are numerous classes ofopioid receptors which bind to exogenous and endogenous opioids. Theseclasses differ in their affinity for various opioid ligands and in theircellular and organ distribution. Moreover, although the differentclasses are believed to serve different physiological functions, thereis substantial overlap of function, as well as of distribution.

In particular, there are at least three known types of opioid receptors,mu (μ), delta (δ), and kappa (κ), to which morphine, the enkephalins,and the dynorphins can bind. These three opioid receptor types are thesites of action of opioid ligands producing analgesic effects. However,the type of pain inhibited and the secondary functions vary with eachreceptor type. The mu receptor is generally regarded as primarilyassociated with pain relief, and drug or other chemical dependence,i.e., addiction and alcoholism.

The human mu opioid receptor, which modulates corticotropin releasinghormone, has been isolated and described in PCT Application WO 95/07983(Mar. 23, 1995) (SEQ ID NO:1) as well as in Chen, Y., Mestek, A.,Hurley, J. A., & Yu, L. (1993) Mol. Pharrnacol. 44, 8-12, and Wang, etal., FEBS letters, (1994)338:217-222. Furthermore, SEQ ID NO:1 anreadily be obtained in GENBANK under accession number L25119. The cDNAherefor contains an open reading frame capable of encoding a protein of400 amino acid residues with 94% sequence similarity to the rat muopioid receptor. Hydropathy analysis of the deduced protein indicatesthe presence of seven hydrophobic domains, typical of G-protein-coupledreceptors. The N-terminus contains five potential N-linked glycosylationsites which remain conserved between the human and the rat mu opioidreceptor. A variant in which Asn-40 is changed to Asp (N40D) is reportedin GENBANK Accession No. U12569.

In the body and brain, heroin is hydrolyzed to morphine, which acts atthe mu opioid receptor and results in an euphoric effect and confers thereinforcing properties of the drug and contributes to development ofaddiction. Heroin addiction can be managed through treatment, primarilymethadone maintenance. However, the biological basis of heroin addictionmay include diversity of gene structure. Such genetic diversity of thehuman mu opioid receptor, and the impact of such diversity on receptorfunction, could contribute to the success or failure of pharmacologicalmanagement. Similar problems with respect to patient response topharmacological treatment could occur in most, if not all addictivediseases, such as heroin addiction, alcohol addiction, or cocaineaddiction to name only a few, or a combination thereof.

Moreover, addiction to opioid drugs, especially heroin, is a majorsocial problem in the United States, and throughout the world. Forexample, recent epidemiological assessments sponsored by the NIH-NIDAand other federal agencies have found that around 2.7 million persons inthe United States have used heroin at some time. Moreover, the numbersof “hardcore” long-term heroin addicts (addiction being defined hereinas self administration of a regular, multiple, daily dose use of ashort-acting opioid, such as heroin, for one year or more, with thedevelopment of tolerance, physical dependence and drug-seeking behavior,a definition codified in the Federal guidelines governingpharmacotherapy using long-acting agents such as methadone or LAAM, andused as the minimal requirement for entry into treatment) are nowestimated to be approximately one million persons. In addition, it hasbeen estimated that around 24 million persons in the United States haveused cocaine for some time, and of that number, approximately onemillion use cocaine regularly, and at least 600,000-700,000 are cocaineaddicts.

In view of the importance of the human mu opioid receptor in the studyof addiction, and the epidemic proportions of drug addiction, especiallyto heroin, alcohol or cocaine, or a combination thereof, in the UnitedStates and throughout the world, and its involvement in theneuroendocrine system, and physiological functions regulated thereby,efforts have been made to investigate whether any polymorphisms in thegene encoding the human mu opioid receptor exist in the population, andwhether such polymorphisms result in a phenotype that has an increasedor decreased susceptibility towards development of addiction toexogenous opioids, such as heroin, or alcohol, cocaine, or otheraddictive drugs. For example, in an article entitled Human mu opioidreceptor gene polymorphisms and vulnerability to substance abuse(Berrettini, W. H., Hoehe, M. R., Ferraro, T. N., DeMaria, P. A., andGottheil, E., Addiction Biolog 2:303-308 (1997)), two polymorphisms inthe human mu opioid receptor gene were reported. One polymorphism (G toT) occurs at nucleotide 175 preceding initiation of translation, and asecond coding polymorphism© to T) at nucleotide 229 (with respect totranscription initiation) on exon I results in an Ala to Val residuechange. However, data taken from a study indicated the C229Tpolymorphism does not differ in occurrence with statistical significancein addicts relative to non addicts (Id at 306). No functional studieswere reported.

It has been further determined that a receptor for both endogenous andexogenous opioids modulates the activity of the hypothalamus pituitaryadrenal axis (HPA) and the hypothalamus pituitary gonadal axis (HPG),which effects the neuroendocrine system and its production of signalingcompounds that play important roles in regulation of numerousphysiological functions. In particular, the neuroendocrine systeminvolves the integration of the neural and endocrine systems of thebody, and is responsible for the coordination of numerous bodilyfunctions. An important part of this system is the hypothalamus, aspecialized portion of the brain involved in receiving and relayingmessages from the central nervous system to other parts of the body.Upon stimulation by chemical signals from the central nervous system,the hypothalamus secretes hypothalamic hormones, such as corticotropinreleasing factor (CRF) or hormone and gonadotropin releasing hormone orluteinizing hormone releasing hormone. These factors in turn stimulatethe anterior pituitary gland to secrete tropic hormones, or tropins,which are synthesized as relatively long polypeptides, and then are thenbiotransformed to produce active peptide hormones. Pro-opiomelanocortin,which is processed into several active peptide hormones, includingadrenocorticotropic hormone (ACTH), is an example of a tropic hormone.ACTH stimulates the adrenal cortex to secrete additional hormones, likecortisol, a stress hormone in humans which regulates glucose metabolism,and targets many tissues in the body. In addition, examples of hormonesproduced by the anterior pituitary glad upon stimulation withgonadotropin releasing hormone include follicle-stimulating hormone andluteinizing hormones. These hormones stimulate the gonads, such as theovaries and the testes, to secrete androgens, such as testosterone,progesterone, and estrogen, which in turn affect sexual development,sexual behavior, and other reproductive and nonreproductive functions.As a result, the endogenous opioid system plays an important role inmodulating endocrine, reproductive, cardiovascular, respiratory,gastrointestinal, immune functions, sexual development and function, aswell as a person's response to stress.

More specifically, in humans, it has been determined that chronicadministration of opioids has an inhibitory effect on the HPA axis[McDonald et al., Effect of morphine and nalorphine on plasmahydrocortisone levels in man. J. Pharmacol. Exp. Ther. 125:241247(1959)]. Basal levels of ACTH and cortisol are significantly disruptedin active heroin addicts: suppression of ACTH and cortisol and abnormaldiurnal rhythms of these hormones are found [Kreek, Medical safety andside effects of methadone in tolerant individuals. JAMA 223:665-668(1973)]. Basal levels and the diurnal rhythm of ACTH and cortisol, whichare disrupted in active heroin addicts, have been shown to becomenormalized in moderate to high dose, long-term methadone-maintainedpatients when compared to those of healthy volunteer subjects [Kreek,1973; Kreek et al., Circadian rhythms and levels of beta-endorphin,ACTH, and cortisol during chronic methadone maintenance treatment inhumans. Life Sci. 33:409-411 (1983); Kreek et al., Prolonged (24 hour)infusion of the opioid antagonist naloxone does not significantly alterplasma levels of cortisol and ACTH in humans. Proceedings of the 7thInternational Congress on Endocrinology Elsevier Science p1170, 1984].

In healthy volunteers, ACTH and cortisol levels decrease below the basallevels in response to the infusion of β-endorphin indicating feedback ofinhibition of pituitary ACTH release or suppression of hypothalamic CRFrelease by β-endorphin [Taylor, et al., Beta-endorphin suppressesadrenocroticotropin and conisol levels in normal human subjects. J.Clin. Endocrinol. Metab. 57:592-596 (1983)], and also naloxone (anopioid antagonist) stimulates a rise in serum ACTH and cortisol,suggesting that the HPA axis is under the tonic inhibitory control ofendogenous opioids normalized in steady-state chronicmethadone-maintained patients; their HPA axis responses tometyrapone-induced stress appear to be no different from that of healthyvolunteer subjects [Kreek, 1973; Kreek et al., Prolonged (24 hour)infusion of the opioid antagonist naloxone does not significantly alterplasma levels of cortisol and ACTH in humans. Proceedings of the 7thInternational Congress on Endocrinology Elsevier Science p1170, 1984].

Support for the effects of opioids on physiological functions regulatedby the HPA and the HPG axes can be found in observations of heroinaddicts. More specifically, it has been observed that many heroinaddicts are infertile, and in the case of female addicts, theirmenstrual cycle is dramatically disrupted to the point that they do notovulate. Furthermore, it has been observed that heroin addicts, andnonaddicted patients taking morphine, become constipated, and that theimmune systems of addicts is weakened relative to the immune system ofnon addicts. However, once therapeutic agents designed to treataddiction, such as methadone, addicts become fertile, are no longerconstipated, and have a immune system whose ability to fight foreignbodies is in parity with the immune system of a nonaddict.

Hence, what is needed is discovery of heretofore unknown polymorphismsof the human mu opioid receptor gene that can be used as genetic markersto map the locus of the human mu opioid receptor gene in the genome.

What is also needed are the DNA sequences of heretofore unknown isolatednucleic acid molecules which encode human mu opioid receptors, whereinthe DNA sequences include a combination of presently known andsubsequently discovered polymorphisms of the human mu opioid receptors.

Furthermore, what is needed is the characterization of the bindingproperties of heretofore unknown human mu opioid receptors produced fromthe expression of genes comprising such heretofore unknown polymorphismsof the human mu opioid receptor gene, or combinations of unknownpolymorphisms and known polymorphisms.

Furthermore, what is needed is a characterization of the activity ofsuch unknown human mu opioid receptors produced from the expression ofnucleic acid molecules comprising such polymorphisms.

What is also needed is a correlation between polymorphisms of the humanmu opioid receptor gene, and the susceptibility of a subject toaddictive diseases, such as heroin addiction, cocaine addiction, oralcohol addiction, to name only a few.

What is also needed are diagnostic methods to determine a subject'sincreased or decreased susceptibility to addictive diseases. With theresults of such methods, targeted prevention methods, early therapeuticintervention, and improved chronic treatment to opioid addiction can bedeveloped. Physicians armed with the results of such diagnostic methodscan determine whether administration to a subject of opioid analgesicsis appropriate or whether non-opioid derived analgesics should beadministered to the subject. Also, appropriate choice and type ofanalgesic can be made in treating a subject's pain.

What is also need are methods of determining a subject's susceptibilityto pain and responsibility to analgesics, and using that informationwhen prescribing analgesics to the subject. What is also needed is anability to determine the binding affinity of the mu opioid receptor toendogenous opioids, such as β-endorphin, and the effect of this bindingactivity on the neuroendocrine system.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

SUMMARY OF THE INVENTION

There is provided, in accordance with the present invention, heretoforeunknown polymorphisms of the human mu opioid receptor gene, and theiruse in mapping the locus of the human mu opioid receptor gene,determining susceptibility to addictive diseases, determiningsusceptibility to pain, and determining a therapeutically effectiveamount of pain reliever to administer to a subject suffering from pain,diagnosing a disease or disorder in a subject that is related to aphysiological function regulated by the HPA or HPG axes of theneuroendocrine system, and selecting an appropriate therapeutic agentand a therapeutically effective amount of such an agent to administer toa subject suffering from a disease or disorder related to aphysiological function regulated by the HPA or HPG.

Hence, the present invention extends to heretofore unknown polymorphismsof the human mu opioid receptor gene that can serve as genetic markersto map the locus of the human mu opioid receptor gene.

The present invention further extends to DNA sequences of heretoforeunknown isolated nucleic acid molecules which encode human mu opioidreceptors, wherein the DNA sequences include a combination of presentlyknown polymorphisms and polymorphisms of the human mu opioid receptorsdiscovered by Applicants.

The present invention further extends to the characterization of thebinding properties of heretofore unknown human mu opioid receptorsproduced from the expression of isolated nucleic acid moleculescomprising DNA sequences with such heretofore unknown polymorphisms ofthe human mu opioid receptor gene, or combinations of unknownpolymorphisms and known polymorphisms.

Furthermore, the present invention extends to characterizing theactivity of such unknown human mu opioid receptors and particularly theincreased or decreased ability of mu opioid receptors produced fromisolated nucleic acid acids of the present invention to activate Gprotein-activated inwardly rectifying K⁺ (GIRK) channels via a Gprotein-mediated mechanism.

The present invention further extends to Applicants' discovery thatpolymorphisms in an allele comprising a DNA sequence of SEQ ID NO:1,such as A118G and C17T, which are described in further detail infra, arepresent in the population at a high frequency (greater than 5%).

Furthermore, the present invention extends to Applicant's discovery of acorrelation between polymorphisms of the human mu opioid receptor gene,and the increased or decreased susceptibility of a subject to addictivediseases, such as heroin addiction, cocaine addiction, or alcoholaddiction, to name only a few.

The present invention further extends to diagnostic methods to determinea subject's increased or decreased susceptibility to addictive diseases.With the results of such methods, targeted prevention methods, earlytherapeutic intervention, and improved chronic treatment to opioidaddiction are set forth herein and encompassed by the present invention.In addition, attending medical professionals armed with the results ofsuch diagnostic methods can determine whether administration of opioidanalgesics is appropriate or whether non-opioid derived analgesicsshould be administered to the subject. Furthermore, appropriate choiceand type of analgesic to treat a subject's pain can be made.

Also, the present invention extends to methods of determining asubject's increased or decreased susceptibility to pain and response toanalgesics, and the use of the information in prescribing analgesics tothe subject.

In addition, the present invention extends to methods of diagnosing adisease or disorder in a subject, wherein the disease or disorder isrelated to a physiological function regulated by the HPA or HPG axes ofthe neuroendocrine system. Examples of such physiological functionsinclude reproductive or sexual functions, gastrointestinal motility,immune response, and ability to withstand stress.

Broadly the present invention extends to an isolated variant allele of ahuman mu opioid receptor gene which can serve as a genetic marker,wherein the predominant or “most common” allele of a human mu opioidreceptor gene found in the population comprises a DNA sequence of SEQ IDNO:1, and a variant allele of the present invention comprises a DNAsequence having a variation in SEQ ID NO:1, wherein the variationcomprises:

G24A;

G779A; or

G942A,

or combinations thereof.

Furthermore, the present invention extends to an isolated variant alleleof a human mu opioid receptor gene as set forth above, which isdetectably labeled. Numerous detectable labels have applications in thepresent invention, such as radioactive elements, chemicals whichfluoresces, or enzymes, to name only a few.

The present invention further extends to an isolated nucleic acidmolecule hybridizable under standard hybridization conditions to anisolated variant allele of the human mu opioid receptor gene, whereinthe predominant or “most common” allele of a human mu opioid receptorgene found in the population comprises a DNA sequence of SEQ ID NO:1,and a variant allele of the present invention comprises a DNA sequencehaving a variation in SEQ ID NO:1, wherein the variation comprises:

G24A;

G779A; or

G942A,

or combinations thereof.

Moreover, the present invention extends to an isolated nucleic acidmolecule hybridizable under standard hybridization conditions to anisolated variant allele of the human mu opioid receptor gene, whereinthe predominant or “most common” allele of a human mu opioid receptorgene found in the population comprises a DNA sequence of SEQ ID NO:1,and a variant allele of the present invention comprises a DNA sequencehaving a variation in SEQ ID NO:1, wherein the variation comprises:

G24A;

G779A; or

G942A,

or combinations thereof, wherein the isolated nucleic acid molecule isdetectably labeled. Examples of detectable labels that have applicationsin this embodiment of the present invention are described above.

In addition, the present invention extends to an isolated variant alleleof a human mu opioid receptor gene, wherein the predominant or “mostcommon” allele of the human mu opioid receptor gene encodes a human muopioid receptor comprising an amino acid sequence of SEQ ID NO:2, andthe variant allele of the human mu opioid receptor gene encodes avariant human mu opioid receptor comprising an amino acid sequencehaving a variation in SEQ ID NO:2, wherein the variation comprisesArg260His.

Furthermore, the present invention extends to an isolated nucleic acidmolecule hybridizable under standard hybridization conditions to anisolated variant allele of a human mu opioid receptor gene of thepresent invention, wherein the isolated nucleic acid molecule encodes avariant human mu opioid receptor comprising an amino acid sequencehaving a variation in SEQ ID NO:2, wherein the variation comprisesArg260His.

Naturally, the present invention extends to a variant human mu opioidreceptor comprising an amino acid sequence having a variation in SEQ IDNO:2, wherein the variation comprises Arg260His.

Furthermore, the present invention extends to an antibody having avariant human mu opioid receptor comprising an amino acid sequencehaving a variation in SEQ ID NO:2, wherein the variation comprisesArg260His as an immunogen. Such an antibody can be a polyclonalantibody, a monoclonal antibody, or a chimeric antibody. Moreover, anantibody of the present invention can be detectably labeled. Examples ofdetectable labels which have applications in this embodiment comprises aradioactive element, a chemical which fluoresces, or an enzyme, to nameonly a few.

In addition, the present invention extends to cloning vectors that canbe used to clone copies of a variant alleles of a human mu opioidreceptor gene of the present invention. For example, the presentinvention extends to a cloning vector comprising an isolated variantallele of a human mu opioid receptor gene and an origin of replication,wherein the predominant or “most common” allele of a human mu opioidreceptor gene found in the population comprises a DNA sequence of SEQ IDNO:1, and a variant allele of the present invention comprises a DNAsequence having a variation in SEQ ID NO:1, wherein the variationcomprises:

G24A;

G779A; or

G942A,

or combinations thereof.

In another embodiment, the present invention extends to a cloning vectorcomprising an isolated nucleic acid molecule hybridizable under standardhybridization conditions to an isolated variant allele of a human muopioid receptor gene, and an origin of replication, wherein thepredominant or “most common” allele of a human mu opioid receptor genefound in the population comprises a DNA sequence of SEQ ID NO:1, and avariant allele of the present invention comprises a DNA sequence havinga variation in SEQ ID NO:1, wherein the variation comprises:

G24A;

G779A; or

G942A,

or combinations thereof.

Numerous cloning vectors have applications in the present invention. Forexample, a cloning vector having applications in the present inventionincludes E. coli, bacteriophages such as lambda derivatives, plasmidssuch as pBR322 derivatives, and pUC plasmid derivatives such as pGEXvectors or pmal-c or pFLAG, to name only a few.

Naturally, the present invention extends to expression vectorscomprising an isolated variant allele a human mu opioid receptor geneoperatively associated with a promoter, wherein the predominant or “mostcommon” allele of a human mu opioid receptor gene found in thepopulation comprises a DNA sequence of SEQ ID NO:1, and a variant alleleof the present invention comprises a DNA sequence having a variation inSEQ ID NO:1, wherein the variation comprises:

G24A;

G779A; or

G942A,

or combinations thereof.

Furthermore, the present invention extends to an expression vectorcomprising an isolated nucleic acid molecule hybridizable under standardhybridization conditions to an isolated variant allele a human mu opioidreceptor gene, wherein the isolated nucleic acid molecule is operativelyassociated with a promoter. As set forth above, the predominant or “mostcommon” allele of a human mu opioid receptor gene found in thepopulation comprises a DNA sequence of SEQ ID NO:1, and a variant alleleof the present invention comprises a DNA sequence having a variation inSEQ ID NO:1, wherein the variation comprises:

G24A;

G779A; or

G942A,

or combinations thereof

Numerous promoters have applications in an expression vector of thepresent invention, including but not limited to immediate earlypromoters of hCMV, early promoters of SV40, early promoters ofadenoviris, early promoters of vaccinia, early promoters of polyoma,late promoters of SV40, late promoters of adenovirus, late promoters ofvaccinia, late promoters of polyoma, the lac the trp system, the TACsystem, the ARC system, the major operator and promoter regions of phagelambda, control regions of fd coat protein, 3-phosphoglycerate kinasepromoter, acid phosphatase promoter, or promoters of yeast a matingfactor, to name only a few.

In addition, the present invention extends to a unicellular hosttransformed or transfected with an expression vector of the presentinvention. Examples of hosts which can be transformed or transfectedwith an expression vector of the present invention, and haveapplications in the present invention, include, but are not limited to,E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, R1.1, B-W,L-M, COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cells.

Naturally, the present invention extends to a method of producing avariant human mu opioid receptor comprising an amino acid sequencehaving a variation in SEQ ID NO:2, wherein the variation comprisesArg260His. An example of such a method comprises the steps of culturinga unicellular host transformed or transfected with an expression vectorcomprising an isolated variant allele a human mu opioid receptor gene,wherein the predominant or “most common” allele of a human mu opioidreceptor gene found in the population comprises a DNA sequence of SEQ IDNO:1, and a variant allele of the present invention comprises a DNAsequence having a variation in SEQ ID NO:1, wherein the variationcomprises G779A, operatively associated with a promoter. The transformedor transfected unicellular host is then cultured under conditions thatprovide for expression of the variant allele of the human mu opioidreceptor gene. The variant human mu opioid receptor produced from suchinduced expression is then recovered from the unicellular host.

Another example comprises the steps of culturing a unicellular hosttransformed or transfected with an expression vector comprising anisolated nucleic acid molecule operatively associated with a promoter,wherein the isolated nucleic acid molecule is hybridizable understandard hybridization conditions to a variant allele a human mu opioidreceptor gene, and the predominant or “most common” allele of a human muopioid receptor gene found in the population comprises a DNA sequence ofSEQ ID NO:1, and the variant allele comprises a DNA sequence having atleast one variation in SEQ ID NO:1, wherein the at least one variationcomprises G779A. The transformed or transfected unicellular host is thencultured under conditions that provide for expression of the variantallele of the human mu opioid receptor gene. The variant human opioidreceptor produced from such induced expression is then recovered fromthe unicellular host.

Furthermore, the present invention extends to an isolated variant alleleof a human mu opioid receptor gene, wherein the predominant or “mostcommon” allele of the human mu opioid receptor gene comprises a DNAsequence of SEQ ID NO:1, and a variant allele of the present inventioncomprises a DNA sequence having at least two variations in SEQ ID NO:1,wherein the variations comprise:

A118G;

C17T;

G24A;

G779A; or

G942A.

The present invention further extends to an isolated variant allele of ahuman mu opioid receptor gone comprising a DNA sequence having at leasttwo variations in SEQ ID NO:1, as stated above, which is detectablylabeled. Examples of detectable labels having applications in thisembodiment include, but are not limited to, a radioactive element, achemical which fluoresces, or an enzyme.

The present invention further extends to an isolated nucleic acidmolecule hybridizable under standard hybridization conditions to anisolated variant allele of a human mu opioid receptor gene, wherein thepredominant or “most common” allele of the human mu opioid receptor genecomprises a DNA sequence of SEQ ID NO:1, and a variant allele of thepresent invention comprises a DNA sequence having at least twovariations in SEQ ID NO:1, wherein the variations comprise:

A118G;

C17T;

G24A;

G779A; or

G942A.

Naturally, the present invention extends to a detectably labeledisolated nucleic acid molecule hybridizable under standard hybridizationconditions to an isolated variant allele of a human mu opioid receptorcomprising a DNA sequence having at least two variations in SEQ ID NO:1,wherein the variations comprise:

A118G;

C17T;

G24A;

G779A; or

G942A.

Examples of detectable labels having applications in this embodiment ofthe invention include, but are not limited to, a radioactive element, achemical which fluoresces, or an enzyme.

Furthermore, the present invention extends to an isolated variant alleleof a human mu opioid receptor gene comprising a DNA sequence having atleast two variations in SEQ ID NO:1, as set forth above, wherein thepredominant or “most common” allele of a human mu opioid receptor geneencodes a human mu opioid receptor comprising an amino acid sequence ofSEQ ID NO:2, and a variant allele of the present invention encodes ahuman mu opioid receptor comprising an amino acid having at least twovariations in SEQ ID NO:2, wherein the variations comprise:

Asn40Asp or conserved variants thereof;

Ala6Val or conserved variants thereof; or

Arg260His or conserved variants thereof

The present invention further extends to an isolated nucleic acidmolecule hybridizable under standard hybridization conditions to anisolated variant allele of a human mu opioid receptor gene comprising aDNA sequence having at least two variations in SEQ ID NO:1, wherein thevariations comprise:

A118G;

C17T;

G24A;

G779A; or

G942A,

such that the isolated nucleic acid molecule encodes a variant human muopioid receptor comprising an amino acid sequence having at least twovariations in SEQ ID NO:2, wherein the variations comprise:

Asn40Asp or conserved variants thereof;

Ala6Val or conserved variants thereof; or

Arg260His or conserved variants thereof.

Naturally, the present invention extends to a variant human mu opioidreceptor comprising an amino acid sequence having at least twovariations in SEQ ID NO:2, wherein the variations comprise:

Asn40Asp or conserved variants thereof;

Ala6Val or conserved variants thereof; or

Arg260His or conserved variants thereof.

Moreover, the present invention extends to an antibody having as animmunogen a human mu opioid receptor comprising an amino acid sequencehaving at least two variations in SEQ ID NO:2, wherein the variationscomprise:

Asn40Asp or conserved variants thereof,

Ala6Val or conserved variants thereof; or

Arg260His or conserved variants thereof.

An antibody of the present invention can be a polyclonal antibody, amonoclonal antibody, or a chimeric antibody. Moreover, an antibody ofthe present invention can be detectably labeled. Examples of detectablelabels having applications in an antibody of the present inventioninclude, but are not limited to, a radioactive element, a chemical whichfluoresces, or an enzyme.

Furthermore, the present invention extends to a cloning vectorcomprising an isolated variant allele of a human mu opioid receptor geneand an origin of replication, wherein the predominant or “most common”allele of the human mu opioid receptor gene present in the populationcomprises a DNA sequence of SEQ ID NO:1, and a variant allele of thepresent invention comprises a DNA sequence having at least twovariations in SEQ ID NO:1, wherein the variations comprise:

A118G;

C17T;

G24A;

G779A; or

G942A.

In addition, the present invention extends to a cloning vectorcomprising an isolated nucleic acid molecule hybridizable under standardhybridization conditions to a variant allele of a human mu opioidreceptor and an origin of replication, wherein the variant allelecomprises a DNA sequence having at least two variations in SEQ ID NO:1,wherein the variations comprise:

A118G;

C17T;

G24A;

G779A; or

G942A,

and an origin of replication.

Numerous cloning vectors have applications in this embodiment of thepresent invention. Examples of such vectors include, but are not limitedto, E. coli, bacteriophages, such as lambda derivatives, plasmids suchas pBR322 derivatives, and pUC plasmid derivatives such as pGEX vectorsor pmal-c or pFLAG, to name only a few.

Naturally, the present invention extends to an expression vectorcomprising an isolated variant allele of a human mu opioid receptor geneoperatively associated with a promoter, wherein such an isolated variantallele comprises a DNA sequence having at least two variations in SEQ IDNO:1, wherein the variations comprise:

A118G;

C17T;

G24A;

G779A; or

G942A.

In addition, the present invention extends to an expression vectorcomprising an isolated nucleic acid molecule operatively associated witha promoter, wherein the isolated nucleic acid molecule is hybridizableunder standard hybridization conditions to an isolated variant allele ofa human mu opioid receptor gene comprising a DNA sequence having atleast two variations in SEQ ID NO:1, wherein the variations comprise:

A118G;

C17T;

G24A;

G779A; or

G942A.

Numerous promoters are available and have applications in an expressionvector of the present invention. Examples of promoters havingapplications include, but are not limited to immediate early promotersof hCMV, early promoters of SV40, early promoters of adenovirus, earlypromoters of vaccinia, early promoters of polyoma, late promoters ofSV40, late promoters of adenovirus, late promoters of vaccinia, latepromoters of polyoma, the lac the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, controlregions of fd coat protein, 3-phosphoglycerate kinase promoter, acidphosphatase promoter, or promoters of yeast α mating factor, to nameonly a few.

Naturally, the present invention extends to a unicellular hosttransformed or transfected with an expression vector of the presentinvention. Examples of unicellular hosts having applications in anembodiment of the present invention include, but are not limited to, E.coli, Pseudonomas, Bacillus, Streptomyces, yeast, WHO, R1.1, B-W, L-M,COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cells.

In another embodiment, the present invention extends to a method forproducing a human mu opioid receptor comprising an amino acid sequencehaving at least two variations in SEQ ID NO:2, wherein the variationscomprise

Asn40Asp or conserved variants thereof;

Ala6Val or conserved variants thereof; or

Arg260His or conserved variants thereof.

More specifically, an example of a method for producing such a human muopioid receptor comprises the steps of culturing a unicellular hosttransformed or transfected with an expression vector comprising anisolated variant allele of a human mu opioid receptor gene operativelyassociated with a promoter, wherein the variant allele comprises a DNAsequence having at least two variations in SEQ ID NO:1, wherein thevariations comprise:

A118G;

C17T;

G24A;

G779A; or

G942A,

under conditions that provide for expression of the isolated variantallele of a human mu opioid receptor gene. After expression, a varianthuman mu opioid receptor is recovered from the unicellular host.

In another example, a method for producing a human mu opioid receptor ofthe present invention comprises the steps of culturing a unicellularhost transformed or transfected with an expression vector comprising anisolated nucleic acid molecule operatively associated with a promoter,wherein the isolated nucleic acid molecule is hybridizable understandard hybridization conditions to an isolated variant allele of ahuman mu opioid receptor gene comprising a DNA sequence having at leasttwo variations in SEQ ID NO:1, wherein the variations comprise:

A118G;

C17T;

G24A;

G779A; or

G942A,

under conditions that provide for expression of the isolated nucleicacid molecule. The variant human mu opioid receptor produced from theexpression is then recovered from the unicellular host.

Moreover, Applicants have discovered that β-endorphin, an endogenousopioid comprising 31 amino acid residues, and binds to the human muopioid receptor, has a binding affinity approximately three timesgreater for a variant human mu opioid receptor produced from expressionof a variant allele of the human mu opioid receptor gene comprising anA118G variation in SEQ ID NO:1, than for a human mu opioid receptorproduced from expression of the predominant or “most common” allele ofthe human mu opioid receptor gene. Also, β-endorphin induced activity ofa receptor produced from the expression of a variant allele of a humanmu opioid receptor gene or an isolated nucleic acid moleculehybridizable thereto under standard hybridization conditions comprisingthe A118G polymorphism effected the receptor's activation of GIRKchannels via a & protein-mediated mechanism relative to the activity ofa receptor produced from the expression of the predominant or “mostcommon” allele. β-endorphin is present in both the central nervoussystem (CNS) and the periphery. It plays a role in endogenous analgesia,as well as in response to exposure to a potential addictive agent, suchas heroin or alcohol. For example, as a neuropeptide, it can modulateneurotransmitter actions in the CNS to mediate antinoception. It is alsoof potential importance for the pathophysiology of addictive diseases.Given the diverse roles of β-endorphin, the presence of a variant alleleof a human mu opioid receptor gene comprising at least one variation inSEQ ID NO:1, wherein the variation comprises A118G, in a subject mayalter the subject's perception of pain, susceptibility to develop opioidaddiction following exposure to opioids as well as addictions to otherdrugs that alter the opioid system, and reaction of the subject towardsa therapeutic agent designed to treat pain, such as morphine, or towardsa therapeutic agent designed to treat a specific addiction. In fact, theinventors herein have also discovered that the A118G variantpolymorphism is present in the human mu opioid receptor gene of at leastone subset of nonaddicts, an Hispanic study subject group, in astatistically significant level (P=0.0041). Hence, the presence of theA118G polymorphism in a subject, with increased β-endorphin affinity andeffects on activation of GIRK channels as described above, is expectedto decrease the subject's perception of pain, protect the subjectagainst potential addiction to opioids such as heroin and likewiseinfluence the subject's reaction to therapeutic agents designed to treatthe at least one addictive disease of the subject.

Furthermore, Applicants have discovered a variant allele of a human muopioid receptor gene comprising a variation in SEQ ID NO:1, wherein thevariation comprises C17T, is present at a statistically significantgreater frequency in the genome of at least one defined subset ofaddicts suffering from at least one addictive disease, than in thegenomes of people not suffering from such a disease. Hence, the presenceof such a variant allele of a human mu opioid receptor gene may alterperception of pain, susceptibility to develop opioid addiction followingexposure to opioids, and influence the subject's reaction to therapeuticagents designed to treat the at least one addictive disease of thesubject. The inventors herein have discovered that the C17T variant muopioid receptor has a endorphin binding affinity approximately 0.72times that of the human mu opioid receptor produced from expression ofthe predominant or “most common” allele of the human mu opioid receptorgene. The decreased binding affinity of this variant is in contrast tothe increased affinity of the A118G variant mentioned above. Inaddition, one of the important indices of cellular functioning of the muopioid receptor is the inhibition of adenylyl cyclase activity followingagonist binding to the receptor. The inventors herein have discoveredthat the C17T variant mu opioid receptor is less sensitive to twoendogenous opioids, β-endorphin and leu-enkephalin, than the receptorproduced by the “most common” allele: the EC50 values differed by afactor of approximately 2.7 and 2.8, respectively. Hence, the presenceof the C17T polymorphism in a subject, with decreased Fendorphinaffinity and decreased sensitivity to endogenous opioids as describedabove, is expected to increase the subject's perception of pain,increase susceptibility to potential addiction to opioids such as heroinand likewise influence the subject's reaction to therapeutic agentsdesigned to treat the at least one addictive disease of the subject.

Accordingly, the present invention extends to a method for determining asusceptibility in a subject to at least one addictive disease,comprising the steps of removing a bodily sample comprising a first andsecond allele of a human mu opioid receptor gene from the subject, anddetermining whether the first allele comprises a human mu opioidreceptor gene comprising a DNA sequence having at least one variation inSEQ ID NO:1, wherein the variation comprises:

A118G; or

C17T.

The present of at least one of these variations in the human mu opioidreceptor gene of the first allele is expected to be indicative of thesubject's susceptibility to at least one addictive disease relative tothe susceptibility of a standard to at least one addictive disease,wherein the standard comprises a first allele comprising a human muopioid receptor gene having a DNA sequence of SEQ ID NO:1.

Another embodiment of the method for determining a susceptibility in thesubject to at least one addictive disease, as described above, comprisesthe further step of determining whether the second allele of the bodilysample of the subject comprises a human mu opioid receptor genecomprising a DNA sequence having at least one variation in SEQ ID NO:1,wherein the variations comprise:

A118G; or

C17T.

The presence of at least one variation the second allele of the bodilysample is expected to be indicative of the subject's susceptibility toat least one addictive disease relative to a standard in which bothalleles of a human mu opioid receptor gene comprise a DNA sequence ofSEQ ID NO:1.

In particular, the presence of an A118G variation in the DNA sequence ofthe human mu opioid receptor gene of the first and/or second alleles inthe bodily sample from the subject is expected to be indicative of adecreased susceptibility of the subject to at least one addictivedisease relative to the standard.

Moreover, the presence of a C17T variation in the DNA sequence of thehuman mu opioid receptor gene of the first and/or second alleles in thebodily sample from the subject is expected to be indicative of anincreased susceptibility of the subject to at least one addictivedisease relative the susceptibility of the standard to at least oneaddictive disease, wherein both alleles of the standard comprise a humanmu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1.Examples of at least one addictive disease includes, but is not limitedto opioid addiction, cocaine addiction or addiction to otherpsychostimulants, nicotine addiction, barbiturate or sedative hypnoticaddiction, anxiolytic addiction, or alcohol addiction.

In another embodiment, the present invention extends to a method fordetermining a susceptibility to at least one addictive disease in asubject relative to susceptibility to at least one addictive disease ina standard, involving the detection of variations in the human mu opioidreceptor itself, and particularly, determining whether a variant humanmu opioid receptor is present in a bodily sample from a subject. Such amethod comprises the steps of removing a bodily sample comprising ahuman mu opioid receptor from the subject, and determining whether thehuman mu opioid receptor present in the sample is a variant human muopioid receptor of the invention, wherein the variant human mu opioidreceptor comprises an amino acid sequence having at least one variationin SEQ ID NO:2, wherein the variation comprises:

Asn40Asp or conserved variants thereof; or

Ala6Val or conserved variants thereof,

The presence of at least one variation is expected to be indicative ofthe subject's susceptibility to at least one addictive disease relativeto susceptibility to at least one addictive disease in a standard,wherein the human mu opioid receptor of the standard comprises an aminoacid sequence of SEQ ID NO:2.

In particular, a variant human mu opioid receptor present in the samplecomprising an amino acid sequence having at least one variation in SEQID NO:2, wherein the variation comprises Asn40Asp or conserved variantsthereof, is expected to be indicative of a decreased susceptibility toat least one addictive disease in the subject relative susceptibility tothe at least one addictive disease in the standard, wherein the human muopioid receptor of the standard comprises an amino acid sequence of SEQID NO:2.

In contrast, a variant human mu opioid receptor present in a sample fromthe subject comprising a variation in SEQ ID NO:2, wherein the variationcomprises Ala6Val or conserved variants thereof, indicates an increasedsusceptibility to addictive diseases in the subject relative to astandard having a human mu opioid receptor comprising an amino acidsequence of SEQ ID NO:2.

As explained above, at least one addictive disease includes, but is notlimited to, opioid addiction, cocaine addiction or addiction to otherpsychostimulants, nicotine addiction, barbiturate or sedative hypnoticaddiction, anxiolytic addiction, or alcohol addiction.

Furthermore, the present invention extends to a method for determining asusceptibility to pain in a subject relative to susceptibility to painin a standard, comprising the steps of removing a bodily samplecomprising a first and second allele of a human mu opioid receptor genefrom the subject, and determining whether the first allele comprises ahuman mu opioid receptor gene comprising a DNA sequence having at leastone variation in SEQ ID NO:1, wherein the variation comprises:

A118G; or

C17T.

The presence of at least one variation in the human mu opioid receptorgene of the first allele is expected to be indicative of a decreased orincreased susceptibility to pain in the subject relative tosusceptibility to pain in the standard, wherein the first allele of thestandard comprises a human mu opioid receptor gene comprising a DNAsequence of SEQ ID NO:1.

Moreover, a method for determining a susceptibility to pain in a subjectmay further comprise the step of determining whether the second allelecomprises a human mu opioid receptor gene comprising a DNA sequencehaving at least one variation in SEQ ID NO:1, wherein the variationcomprises:

A118G; or

C17T.

The presence of the at least one variation in the human mu opioidreceptor gene of the second allele of the bodily sample from the subjectis expected to be indicative of an increased or decreased susceptibilityto pain in the subject relative to the susceptibility to pain in thestandard, wherein the second allele in the standard comprises a human muopioid receptor gene comprising a DNA sequence of SEQ ID NO:1.

At least one variation in the human mu opioid receptor gene of the firstand/or second allele of the bodily sample taken from the subject,wherein the variation comprises A118G, is expected to be indicative of adecreased susceptibility to pain in the subject relative susceptibilityof pain in the standard, wherein the human mu opioid receptor gene ofthe first and/or second allele of the standard comprises a DNA sequenceof SEQ ID NO:1.

Furthermore, the presence of at least one variation comprising C17T inthe human mu opioid receptor gene of the first and/or second allele ofthe bodily sample from the subject is expected to be indicative of anincreased susceptibility to pain in the subject relative to thesusceptibility to pain in the standard, wherein the first and/or secondallele of the standard comprise a human mu opioid receptor genecomprising a DNA sequence of SEQ ID NO:1.

In another embodiment, the present invention extends to a method fordetermining a susceptibility to pain in a subject relative tosusceptibility to pain in a standard by examining a bodily sample takenfrom the subject for the presence of a variant human mu opioid receptor.Such a method comprises the steps of removing a bodily sample comprisinga human mu opioid receptor from the subject, and determining whether thehuman mu opioid receptor present in the sample is a variant human muopioid receptor of the invention, i.e., comprises an amino acid sequencehaving at least one variation in SEQ ID NO:2, wherein the variationcomprises:

BAsn40Asp or conserved variants thereof; or

Ala6Val or conserved variants thereof,

such that the presence of at least one variation is expected to beindicative of the subject's susceptibility to pain relative tosusceptibility to pain in the standard, wherein the human mu opioidreceptor of the standard comprises an amino acid sequence of SEQ IDNO:2.

In particular, the presence of a variant human mu opioid receptorcomprising an amino acid sequence having at least one variation in SEQID NO:2 wherein the variation comprises Asn40Asp or conserved variantsthereof, is expected to be indicative of a decreased susceptibility topain in the subject relative to susceptibility to pain in the standard,wherein the human mu opioid receptor of the standard comprises an aminoacid sequence of SEQ ID NO:2.

Furthermore, the presence of a variant human mu opioid receptorcomprising an amino acid sequence having a variation in SEQ ID NO:2,wherein the variation comprises Ala6Val or conserved variants thereof,in a bodily sample taken from a subject is expected to be indicative ofan increased susceptibility to pain in the subject relative tosusceptibility to pain in the standard, wherein the human mu opioidreceptor of the standard comprises an amino acid sequence of SEQ IDNO:2.

Once a susceptibility to pain in the subject has been determined, it ispossible for attending medical professionals treating the subject toadminister to an appropriate, or therapeutically effective amount ofpain reliever in order to induce analgesia in the subject.Administration of such an amount is important to the subject because,should an inappropriate amount of pain reliever be administered, thesubject may not experience analgesia, and may be exposed to potentiallydeleterious side effects of the pain reliever, such as induction ofaddiction to the pain reliever, brain damage, or death.

Consequently, the present invention extends to a method for determininga therapeutically effective amount of pain reliever to administer to asubject in order to induce analgesia in the subject relative to atherapeutically effective amount of the pain reliever to administer to astandard in order to induce analgesia in the standard, wherein themethod comprises determining a susceptibility to pain in the subjectrelative to susceptibility to pain in the standard. The susceptibilityof pain in the subject is expected to be indicative of thetherapeutically effective amount of the pain reliever to administer tothe subject to induce analgesia in the subject relative to the amount ofthe pain reliever to administer to the standard to induce analgesia inthe standard.

Hence, the present invention extends to a method for determining atherapeutically effective amount of pain reliever to administer to asubject in order to induce analgesia in the subject relative to atherapeutically effective amount of the pain reliever to administer to astandard in order to induce analgesia in the standard wherein the methodcomprises the steps of removing a bodily sample comprising a first andsecond allele of a human mu opioid receptor gene from the subject, anddetermining whether the first allele comprises a human mu opioidreceptor gene comprising a DNA sequence having at least one variation inSEQ ID NO:1, wherein the variation comprises:

A118G; or

C17T.

The presence of at least one variation in the human mu opioid receptorgene of the first allele from the bodily sample is expected to beindicative of the therapeutically effective amount of pain reliever toadminister to the subject to induce analgesia in the subject relative tothe therapeutically effective amount of pain reliever to administer tothe standard to induce analgesia in the standard, wherein the standardcomprises a first allele comprising a human mu opioid receptor genecomprising a DNA sequence of SEQ ID NO:1.

Moreover, the present invention further extends to a method fordetermining a therapeutically effective amount of pain reliever toadminister to a subject in order to induce analgesia in the subjectrelative to a therapeutically effective amount of pain reliever toadminister to a standard to induce analgesia therein, further comprisingthe steps of removing a bodily sample comprising a first and secondallele comprising a human mu opioid receptor gene from the subject, anddetermining whether the second allele of the bodily sample comprises ahuman mu opioid receptor gene comprising a DNA sequence comprising atleast one variation in SEQ ID NO:1, wherein the variation comprises:

A118G; or

C17T.

The presence of at least one variation in the human mu opioid receptorgene of the first and/or second allele of the bodily sample is expectedto be indicative of the therapeutically effective amount of painreliever to administer to the subject to induce analgesia thereinrelative to the amount of pain reliever to administer to a standard toinduce analgesia therein, wherein the first and second alleles of thestandard comprise a human mu opioid receptor gene comprising a DNAsequence of SEQ ID NO:1.

More particularly, a variation of the human mu opioid receptor gene ofthe first and/or second allele from the bodily sample taken from thesubject, comprising a DNA sequence comprising a variation in SEQ IDNO:1, wherein the variation comprises A18G, is expected to be indicativeof a decreased susceptibility to pain in the subject relativesusceptibility of pain in the standard. Consequently, the subjectrequires a decreased therapeutically effective amount of pain relieverin order to induce analgesia therein relative to the therapeuticallyeffective amount of pain reliever needed to induce analgesia in thestandard.

In contrast, a variation of the DNA sequence of the human mu opioidreceptor gene of the first and/or second allele from the bodily sampletaken from the subject, comprising C17T, is expected to be indicative ofan increased susceptibility to pain in the subject relative to thesusceptibility to pain in the standard. Hence, the therapeuticallyeffective amount of pain reliever to administer to the subject in orderto induce analgesia therein is greater than the therapeuticallyeffective amount of pain reliever to administer to the standard toinduce analgesia therein.

In another embodiment, the present invention extends to determining atherapeutically effective amount of pain reliever to administer to asubject in order to induce analgesia in the subject, by examining abodily sample from a subject for the presence of a variant human muopioid receptor comprising an amino acid sequence having a variation inSEQ ID NO:2. More specifically, the present invention extends to amethod for determining a therapeutically effective amount of painreliever to administer to a subject in order to induce analgesia in thesubject, relative to a therapeutically effective amount of pain relieverto administer to a standard in order to induce analgesia in thestandard, comprising the steps of removing a bodily sample comprising ahuman mu opioid receptor from the subject, and determining whether thehuman mu opioid receptor present in the sample comprises an amino acidsequence having at least one variation in SEQ ID NO:2, wherein thevariation comprises:

Asn40Asp or conserved variants thereof; or

Ala6Val or conserved variants thereof,

such that the presence of at least one variation is expected to beindicative of the therapeutically effective amount of pain reliever toadminister to the subject to induce analgesia therein relative to thetherapeutically effective amount of pain reliever to administer toinduce analgesia in the standard, wherein the human mu opioid receptorof the standard comprises an amino acid sequence of SEQ ID NO:2.

In particular, the presence of a variant human mu opioid receptorcomprising an amino acid sequence having a variation in SEQ ID NO:2,wherein the variation comprises Asn40Asp or conserved variants thereofin the sample from the subject, is expected to be indicative of adecreased therapeutically effective amount of pain reliever toadminister to the subject to induce analgesia therein relative to thetherapeutically effective amount of pain reliever to administer to thestandard in order to induce analgesia therein.

In contrast, the presence of a variant human mu opioid receptor in thesample from the subject, wherein the receptor comprises an amino acidsequence have a variation in SEQ ID NO:2, wherein the variationcomprises Ala6Val or conserved variants thereof, is expected to beindicative of an increased therapeutically effective amount of painreliever to administer to the subject in order to induce analgesiatherein relative to the therapeutically effective amount to administerto the standard to induce analgesia therein.

Examples of pain relievers having applications in this embodiment of thepresent invention include, but are not limited to, morphine, codeine,dihydromorphin, meperidine, methadone, fentanyl and its congeners,butorphenol, nalbuphine, LAAM, or propoxyphine, to name only a few.

Furthermore, the present invention extends to a method for determining atherapeutically effective amount of a therapeutic agent for treating atleast one addictive disease to 15 administer to a subject suffering fromat least one addictive disease, relative to a therapeutically effectiveamount of the therapeutic agent to administer to a standard sufferingfrom the at least one addictive disease. As a result, the dosage oftherapeutic agent administered to an addict can be “tailored” to theaddict's needs based upon the addict's genotype. An example of such amethod comprises the steps of removing a bodily sample from the subject,wherein the bodily sample comprises a first and second allele of thehuman mu opioid receptor gene, and determining whether the first allelecomprises a DNA sequence having at least one variation in SEQ ID NO:1,wherein the variation comprises:

A118G; or

C17T.

The presence of the at least one variation in the human mu opioidreceptor gene of the first allele in the bodily sample from the subjectis related to the therapeutically effective amount of therapeutic agentto administer to the subject to treat the subject's at least oneaddictive disease, relative to the therapeutically effective amount ofthe therapeutic agent to administer to the standard suffering from theat least one addictive disease, wherein the first and second allele ofthe standard comprise a human mu opioid receptor gene comprising a DNAsequence of SEQ ID NO:1.

Furthermore, a method for determining a therapeutically effective amountof therapeutic agent to administer to a subject suffering from at leastone addictive disease may further comprise an additional step ofdetermining whether the second allele of the bodily sample taken fromthe subject comprises a human mu opioid receptor gene comprises a DNAsequence having at least one variation in SEQ ID NO:1, wherein the atleast one variation comprises:

A118G; or

C17T.

Such a variation in the first and/or second allele of the bodily sampleis expected to be indicative of the therapeutically effective amount ofthe therapeutic agent to administer to the subject to treat the at leastone addictive disease of the subject relative to the therapeuticallyeffective amount of the therapeutic agent to administer to the standardsuffering from the at least one addictive disease.

The presence of a human mu opioid receptor gene comprising a DNAsequence having at least one variation in SEQ ID NO:1 in the firstand/or second alleles of the bodily sample taken from the subject,wherein the variation comprises A118G is expected to be indicative of adecreased therapeutically effective amount of the therapeutic agent toadminister to the subject to treat the at least one addictive disease ofthe subject, relative to the therapeutically effective amount of thetherapeutic agent to administer to the standard suffering from the atleast one addictive disease, wherein the two alleles of the standardcomprise a human mu opioid receptor gene comprising a DNA sequence ofSEQ ID NO:1.

Furthermore, the presence of a human mu opioid receptor gene comprisinga DNA sequence having at least one variation of SEQ ID NO:1 in the firstand/or second allele of the bodily sample taken from the subject,wherein the variation comprises C17T, is expected to be indicative of anincreased therapeutically effective amount of the therapeutic agent toadminister to treat the at least one addictive disease of the subjectrelative to the therapeutically effective amount of the therapeuticagent to administer to the standard suffering from the at least oneaddictive disease to treat the at least one addictive disease in thestandard, wherein the alleles of the standard comprise a human mu opioidreceptor gene comprising a DNA sequence of SEQ ID NO:1.

In another embodiment, the present invention extends to determining atherapeutically effective amount of a therapeutic agent for treating atleast one addictive disease to administer to a subject suffering from atleast one addictive disease by examining a bodily sample from a subjectfor the presence of a variant human mu opioid receptor comprising anamino acid sequence having at least one variation in SEQ ID NO:2. Morespecifically, the present invention extends to a method for determininga therapeutically effective amount of therapeutic agent for treating atleast one addictive disease to administer to a subject suffering fromthe at least one addictive disease, relative to a therapeuticallyeffective amount of the therapeutic agent to administer to a standardsuffering from the at least one addictive disease, wherein the methodcomprises the steps of removing a bodily sample comprising a human muopioid receptor from the subject, and determining whether the human muopioid receptor present in the sample comprises an amino acid sequencehaving at least one variation in SEQ ID NO:2, wherein the variationcomprises:

Asn40Asp or conserved variants thereof; or

Ala6Val or conserved variants thereof.

The presence of at least one variation in the human mu opioid receptorof the bodily sample is expected to be indicative of therapeuticallyeffective amount of the therapeutic agent to administer to the subjectto treat the at least one addictive disease of the subject relative tothe therapeutically effective amount of the therapeutic agent toadminister to the standard suffering from the at least one addictivedisease, wherein the human mu opioid receptor of the standard comprisesan amino acid sequence of SEQ ID NO:2.

In particular, the presence of a variant human mu opioid receptorcomprising an amino acid sequence having at least one variation in SEQID NO:2 comprising Asn40Asp or conserved variants thereof in the bodilysample of the subject is expected to be indicative of a decreasedtherapeutically effective amount of the therapeutic agent to administerto the subject to treat the at least one addictive disease in thesubject relative to the therapeutically effective amount of thetherapeutic agent to administer to the standard to treat the at leastone addictive disease therein.

Furthermore, the presence of a variant human mu opioid receptorcomprising an amino acid sequence having at least one variation in SEQID NO:2, wherein the variation comprises Ala6Val or conserved variantsthereof in the bodily sample of the subject is expected to be indicativeof an increased therapeutically effective amount of the therapeuticagent to administer to the subject in order treat the at least oneaddictive disease in the subject relative to the therapeuticallyeffective amount of the therapeutic agent to administer to the standardsuffering from the at least one addictive disease. Examples of at leastone addictive disease includes, but is not limited to opioid addiction,cocaine addiction or addiction to other psychostimulants, nicotineaddiction, barbiturate or sedative hypnotic addiction, anxiolyticaddiction, or alcohol addiction. Furthermore, examples of therapeuticagents having applications of the present invention include methadone,LAAM, maltrexone, or buprinorphine, to name only a few.

Furthermore, the present invention extends to a method for diagnosing adisease or disorder related to a physiological function regulated by theHPA or HPG axes of the neuroendocrine system. The HPA and HPG axes playan important role in regulation of numerous physiological activitiessuch as reproductive and sexual function, gastrointestinal motility,immune response to an antigen, or an ability to withstand stress.Furthermore, the HPA and HPG axes exert such regulatory control via theproduction of endogenous opioids that interact with opioid in manylocations of the body. In particular, in the HPG axis, the mu opioidreceptor is centrally involved in tonic regulation of the luteinizinghormone, particularly in its pulsatile release. Furthermore, in the HPAaxis, the mu opioid receptor modulates corticotropin releasingfactor/hormone (CRF or CRH) in the hypothalamus which in turn modulatesproduction of pro-opiomelanocortin (POMC) in the pituitary which isprocessed into several active peptides such as ACTH, which stimulatesthe adrenal cortex to release the stress hormone cortisol in humans,which in turn provides the stress response to environmental stimuli.Furthermore, modulated mu opioid receptor activity can lead tomodulation of most cellular and humoral immunity including that mediatethrough T cells, B cells, cytokines, and chemokines. The pathophysiologyof immune disorders may therefore be influenced by pharmacotherapiesthat modulate the activity of the mu opioid receptor. Moreover,gastrointestinal motility is modulated by modulation of opioid receptortreatment, and diagnosis of a disease or disorder related togastrointestinal motility (e.g. constipation) may be facilitated byknowledge of intrinsic mu opioid receptor motility.

Applicants have discovered that the binding affinity of an opioidreceptor, such as a mu opioid receptor with an endogenous opioid ligand,such as β-endorphin, is expected to modulate such physiologicalactivities. Hence, the binding affinity of variant mu opioid receptorsexplained above, for endogenous opioid ligands such as β-endorphin, isexpected to modulate those physiological activities regulated by the HPAand HPG axes relative to those physiological activities in a standardhaving mu opioid receptors produced from the predominant or “mostcommon” allele of the mu opioid receptor gene comprising a DNA sequenceof SEQ ID NO:1. As the result, the present invention extends to a methodof diagnosing a disease or disorder related to a physiological functionregulated by the HPA or HPG axes. Examples of physiological functionsregulated by the HPA and the HPG include, but are not limited to sexualor reproductive functions, gastrointestinal motility, immune response,or ability to withstand stress. Such a method comprises the steps ofremoving a bodily sample comprising a first and second allele of a humanmu opioid receptor gene from the subject, and determining whether thefirst allele comprises a human mu opioid receptor gene comprising a DNAsequence having at least one variation in SEQ ID NO:1, wherein thevariation comprises:

A118G; or

C17T.

The presence of at least one variation in the human mu opioid receptorgene of the first allele is expected to be indicative of a disorderrelated to a physiological function regulated by the HPA or GPA, such assexual or reproductive functions, gastrointestinal motility, immuneresponse, and the ability to withstand stress, wherein the first alleleof the standard comprises a human mu opioid receptor gene comprising aDNA sequence of SEQ ID NO:1.

Moreover, a method for diagnosing a disease or disorder related to aphysiological function regulated by the HPA or GPA, as described abovemay further comprise the step of determining whether the second alleleof the bodily sample comprises a human mu opioid receptor genecomprising a DNA sequence having at least one variation in SEQ ID NO:1,wherein the variation comprises:

A118G; or

C17T.

The presence of the at least one variation in the human mu opioidreceptor gene of the second allele of the bodily sample from the subjectmay be expected to be indicative of a disease or disorder related tosexual and reproductive functions, gastrointestinal motility, immuneresponse, or the ability of the subject to withstand stress.

At least one variation in the human mu opioid receptor gene of the firstand/or second allele of the bodily sample taken from the subject,wherein the variation comprises A118G, is expected to be indicative ofdecreased HPA and HPG activity, resulting in increased sexual orreproductive functions, increased gastrointestinal motility, increasedimmune response, or increased ability to withstand stress relative tothe levels of such function observed in a standard.

Furthermore, the presence of at least one variation comprising C17T inthe human mu opioid receptor gene of the first and/or second allele ofthe bodily sample from the subject is expected to be indicativeincreased HPA or HPG activity, resulting in decreased sexual orreproductive function, decreased gastrointestinal motility, decreasedimmune response, or decreased ability to withstand stress relative tothe levels of such function observed in a standard.

In another embodiment, the present invention extends to a method fordiagnosing a disease or disorder related to a physiological functionregulated by the HPA or GPA by examining a bodily sample taken from thesubject for the presence of a variant human mu opioid receptor Such amethod comprises the steps of removing a bodily sample comprising ahuman mu opioid receptor from the subject, and determining whether thehuman mu opioid receptor present in the sample is a variant human muopioid receptor of the invention, i.e., comprises an amino acid sequencehaving at least one variation in SEQ ID NO:2, wherein the variationcomprises:

Asn40Asp or conserved variants thereof; or

Ala6Val or conserved variants thereof,

such that the presence of at least one variation is expected to beindicative of a disease or disorder related to a physiological activityregulated by the HPA or HPG axes, such as sexual function ordevelopment, gastric motility, immune response, or the ability of thesubject to withstand stress, relative to regulation of such activitiesin a standard comprising a human mu opioid receptor having an amino acidsequence of SEQ ID NO:2.

In particular, the presence of a variant human mu opioid receptorcomprising an amino acid sequence having at least one variation in SEQID NO:2 wherein the variation comprises Asn40Asp or conserved variantsthereof, is expected to be indicative decreased HPA and HPG activity,resulting in increased sexual or reproductive functions, increasedgastrointestinal motility, increased immune response, or increasedability to withstand stress relative to the levels of such functionobserved in a standard having a mu opioid receptor comprising an aminoacid sequence of SEQ ID NO:2.

Furthermore, the presence of a variant human mu opioid receptorcomprising an amino acid sequence having a variation in SEQ ID NO:2,wherein the variation comprises Ala6Val or conserved variants thereof,in a bodily sample taken from a subject is expected to be indicative ofincreased activity of the HPA and HPG axes, resulting in decreasedsexual or reproductive functions, decreased gastrointestinal motility,decreased immune response, or decreased ability to withstand stressrelative to the levels of such function observed in a standard having amu opioid receptor comprising an amino acid sequence of SEQ ID NO:2.Examples of specific diseases or disorders related to regulation ofphysiological functions by the HPA or HPG axes include infertility,constipation, diarrhea, decreased immune response to antigens relativeto a standard, or decreased of ability to withstand stress relative to astandard.

Once a disease or disorder related to a physiological function regulatedby the HPA or HPG axes has been diagnosed, it is possible for attendingmedical professionals treating the subject to select and administer anappropriate therapeutic agent and a therapeutically effective amount ofthe agent to administer to the subject to treat such a disease ordisorder. Consequently, the present invention extends to a method fordetermining an appropriate therapeutic agent to administer to a subjectsuffering from a disease or disorder related to a physiological functionregulated by the HPA or HPG axes, comprising removing a bodily samplefrom the subject, and determining the presence of at least one variantallele of a mu opioid receptor gene in the bodily sample, wherein thevariant allele comprises a human mu opioid receptor gene comprising aDNA sequence having at least one variation in SEQ ID NO:1, wherein thevariation comprises:

A118G; or

C17T.

The present invention further extends to a method for selecting anappropriate therapeutic agent to administer to a subject suffering froma disease or disorder related to a physiological function regulated bythe HPA or HPG axes as set forth above, further comprising determiningwhether the bodily sample comprises a second variant allele of the muopioid receptor gene comprising a DNA sequence having a variation in SEQID NO:1, wherein the variation comprises:

A118G; or

C17T.

In particular, should either or both alleles of the mu opioid receptorgene of the bodily sample comprise a DNA sequence having a variation inSEQ ID NO:1, wherein the variation comprises A118G, the mu opioidreceptors of the subject are expected to have increased functionrelative to mu opioid receptors of a standard produced from expressionof the predominant or “most common” mu opioid receptor allele comprisinga DNA sequence of SEQ ID NO:1. This increased function is expected toresult in decreased function of the HPA and HPG axes. Hence, anappropriate therapeutic agent for treating a disease or disorder relatedto decreased activity of the HPA or HPG axes, such as diarrhea can beselected.

In contrast, a human mu opioid receptor produced from expression of avariant allele of a mu opioid receptor gene comprising a variation inSEQ ID NO:1, wherein the variation comprises C17T is expected to havedecreased activity relative to a mu opioid receptor produced fromexpression of the predominant or “most common” allele of the human muopioid receptor gene comprising a DNA sequence of SEQ ID NO:1. Thisdecreased activity is expected to result in higher activity of the HPAand HPG axes. As a result, a medical professional attending the subjectis able to select an appropriate therapeutic agent for treating adisease or disorder related to sexual and reproductive functions, suchas infertility, gastrointestinal motility, such as constipation ordiarrhea, decreased immune response towards antigens relative to immuneresponse in a standard, or decreased ability to withstand stressrelative to ability to withstand stress in a standard.

The present invention further extends to commercial test kits suitablefor use by a medical professional to determine whether either or bothalleles of a bodily sample taken from a subject comprise a DNA sequencehaving at least one variation in SEQ ID NO:1, wherein the variationcomprises:

A118G; or

C17T.

Commercial test kits of the present invention have applications indetermining susceptibility of pain in the subject relative to astandard. Such kits can also be used to determine a subject's increasedor decreased susceptibility to at least one addictive disease relativeto susceptibility to at least one addictive disease in a standard. Alsoa therapeutically effective amount of pain reliever to administer to thesubject in order to induce analgesia in the subject relative to atherapeutically effective amount of pain reliever to administer to astandard to induce analgesia in the standard can be determined.Moreover, a test kit of the present invention has applications indetermining a therapeutically effective amount of therapeutic agent fortreating at least one addictive disease to administer to a subjectsuffering from the at least one addictive disease, relative to atherapeutically effective amount of therapeutic agent to administer to astandard suffering from at least one addictive disease. Furthermore,test kits of the invention have applications in diagnosing a disease ordisorder related to a physiological condition regulated by the HPA orHPG axes of the neuroendocrine system, and in selecting an appropriatetherapeutic agent for treating such a disease or disorder, along with atherapeutically effective amount of agent to administer to the subject.A standard as used herein comprises two alleles of a human mu opioidreceptor gene comprising a DNA sequence of SEQ ID NO:1.

Furthermore, a commercial test kit of the present invention can also beused to determine the presence of an isolated variant allele of a humanmu opioid receptor gene of the present invention in a bodily sampleremoved from a subject, which can serve as a genetic marker. Asexplained above, the predominant or “most common” allele of a human muopioid receptor gene found in the population comprises a DNA sequence ofSEQ ID NO:1. Hence a variant allele comprising a DNA sequence having avariation in SEQ ID NO:1, wherein the variation comprises:

G24A;

G779A; or

G942A,

or combinations thereof, can be detected in the bodily sample with acommercial kit of the invention.

Other variant alleles of the human mu opioid receptor gene of thepresent invention can be detected with a commercial test kit of thepresent invention. For example, an isolated variant allele of a human muopioid receptor gene detectable with a commercial kit of the presentinvention, comprises a DNA sequence having at least two variations inSEQ ID NO:1, wherein the variations comprise:

A118G;

C17T;

G24A;

G779A; or

G942A.

Accordingly, a commercial test kit may be prepared for determining thepresence of at least one variation in a human mu opioid receptor gene ofeither or both alleles in a bodily sample taken from a subject, whereinthe commercial test kit comprises:

a) PCR oligonucleotide primers suitable for detection of an allelecomprising a human mu opioid receptor gene having a DNA sequence with avariation in SEQ ID NO:1;

b) other reagents; and

c) directions for use of the kit.

The present invention further extends to commercial test kits capable ofdetecting a variant human mu opioid receptor in a bodily sample takenfrom a subject. Examples of variant human mu opioid receptors that canbe detected with a kit of the present invention comprise:

a variant human mu opioid receptor comprising an amino acid sequencehaving a variation in SEQ ID NO:2, wherein the variation comprisesArg260His or conserved variants thereof; or

a variant human mu opioid receptor comprising an amino acid sequencehaving at least two variations in SEQ ID NO:2, wherein the variationscomprise:

Asn40Asp or conserved variants thereof,

Ala6Val or conserved variants thereof; or

Arg260His or conserved variants thereof.

Moreover, a commercial test kit of the present invention can be used todetermine: susceptibility to pain in the subject relative tosusceptibility to pain in a standard; a therapeutically effective amountof pain reliever to administer to a subject to induce analgesia in thesubject relative to a therapeutically effective amount of pain relieverto administer to a standard to induce analgesia in the standard; atherapeutically effective amount of therapeutic agent for treating atleast one addictive disease to administer to a subject suffering from atleast one addictive disease, relative to a therapeutically effectiveamount of therapeutic agent to administer to a standard suffering fromthe at least one addictive disease; diagnosing a disease or disorderrelated to a physiological condition regulated by the HPA or HPG axes ofthe neuroendocrine system, or selecting an appropriate therapeutic agentfor treating such a disease or disorder, along with a therapeuticallyeffective amount of such agent to administer to the subject.Accordingly, the present invention extends to a commercial test kithaving applications set forth above, comprising a predetermined amountof at least one detectably labeled immunochemically reactive componenthaving affinity for a variant human mu opioid receptor;

(b) other reagents; and

(c) directions for use of the kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. “competitive,” “sandwich,” “double antibody,” etc.), andcomprises:

(a) a labeled component which has been obtained by coupling the human muopioid receptor of a bodily sample to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand comprises:

(i) a ligand capable of binding with the labeled component (a);

(ii) a ligand capable of binding with a binding partner of the labeledcomponent (a);

(iii) a ligand capable of binding with at least one of the component(s)to be determined; or

(iv) a ligand capable of binding with at least one of the bindingpartners of at least one of the component(s) to be determined; or

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between the human mu opioid receptor gene of the presentinvention and a specific binding partner thereto.

Accordingly, it is an object of the present invention to provideheretofore unknown variations the DNA sequence of the human mu opioidreceptor gene wherein the variations can be used to map the locus of thehuman mu opioid receptor gene.

It is yet another object of the present invention to use heretoforeunknown polymorphisms of an allele of the human mu opioid receptor geneas markers for any kind of disorder related to the human mu opioidreceptor, such as an addictive disease, pain, or markers for genes.

It is another object of the present invention to provide nucleotides,optionally detectably labeled, hybridizable under standard hybridizationconditions to variant alleles of the human mu opioid receptor genedisclosed herein, as well as polypeptides produced from the expressionof the variant alleles and nucleotides hybridizable thereto understandard hybridization conditions.

It is yet another object of the present invention to provide antibodies,optionally detectably labeled, having immunogens comprising polypeptidesproduced from the expression of variant alleles of human mu opioidreceptor gene, or expression of isolated nucleic acid moleculeshybridizable under standard hybridization conditions to variant allelesdisclosed herein.

It is another object of the present invention to gain insight into asubject's susceptibility to pain. This insight can be used to determinea therapeutically effective dose of pain reliever to administer to thesubject to induce analgesia therein relative to the therapeuticallyeffective amount of pain reliever administered to a standard to induceanalgesia therein, wherein the standard comprises two alleles of thehuman mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1,or a variant human mu opioid receptor comprising an amino acid sequenceof SEQ ID NO:2.

Such information can be used to tailor a regimen for treating a subjectsuffering from at least one addictive disease, relative to thetherapeutically effective amount of therapeutic agent administered to astandard suffering from at least one addictive disease.

It is yet another object of the present invention to provide commercialtest kits for attending medical professionals to determine the presenceof variant alleles of a human mu opioid receptor gene in a bodily sampletaken from a subject. The results of such testing can then be used todetermine the subject's susceptibility to pain, susceptibility to atleast one addictive disease, determining a therapeutically effectiveamount of pain reliever to administer to the subject in order to induceanalgesia, or determining a therapeutically effective amount oftherapeutic agent for treating at least one addictive disease toadminister to the subject.

It is an object of the present invention to determine the activity of amu opioid receptor in a subject, and use such information to diagnose adisease or disorder related to sexual or reproductive function,gastrointestinal motility, immune response, or ability to withstandstress, wherein variant alleles of the mu opioid receptor gene whenexpressed produce variant mu opioid receptors having activity differentfrom a mu opioid receptor produced from the predominant or “most common”allele of the mu opioid receptor comprising a DNA sequence of SEQ IDNO:1.

It is another object of the present invention to employ Applicants'discovery of a correlation between the activity of a mu opioid and itsimpact the neuroendocrine system, and particularly on levels of hormoneswithin the body. As a result, the level of activity of the mu opioidreceptor effects sexual or reproductive function, gastrointestinalmotility, immune response, or ability to withstand stress. Suchinformation can further be used select appropriate therapeutic agents totreat diseases such as infertility, constipation, or diarrhea. Further,such information can be used to select appropriate therapeutic agents toincrease immune response against an antigen such as a bacterium, a virusor a tumor cell in the subject, and to treat psychiatric diseases ordisorders such as obsessive compulsive disorder, schizophrenia, ordepression.

It is yet another object of the present invention to provide commercialdetecting variant alleles of the human mu opioid receptor gene or thepresence of a variant human mu opioid receptor in a bodily sample takenfrom a subject. The results of such tests can then be used to gainincite into a subject's ability to withstand pain, susceptibility toaddiction, to diagnose a disease or disorder related to a physiologicalfunction regulated by the HPA or HPG axes such as sexual andreproductive functions, gastrointestinal motility, immune response, andthe ability of the subject to withstand stress.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: DNA sequence of the A118G polymorphismn. Examples of DNAsequences are shown from individuals homozygous for the most commonallele (left) (SEQ ID NO:1), heterozygous (center) and homozygous forthe A118G variant allele (right). The arrows indicate the position ofnucleotide 118, with the adjacent sequences shown.

FIG. 2: Binding of endogenous opioid peptides to the most common (SEQ IDNO:2) and Asn40Asp mu opioid receptors. Membrane preparations from cellsexpressing either the most common (open circles) or the A118G variant(filled squares) receptors were used in binding experiments to displacethe [3H]-DAMGO binding. Shown are examples of displacement binding forfour endogenous peptides: Met-enkephalin, dynorphin A, β-endorphin, andendomorphin-1.

FIGS. 3A-3C: Functional comparison of the most common (SEQ ID NO:1) andA118G variant human mu opioid receptors in coupling to Gprotein-activated inwardly rectifying K⁺ (GIRK) channels. (A) Example ofcurrent trace showing the experimental protocol and calculation methodfor the agonist-induced response. Oocytes were clamped at a holdingpotential of −80 mV and superfused with different solutions asindicated. I_(max): maximum K⁺ currents evoked by DAMGO at a saturatingconcentration (100 nM). I_(test): K⁺ currents evoked by the test dose ofagonists. (B) and (C) Dose response curves of receptor activation. Thetested concentrations of agonists ranged from 0.1 nM to 1 μM. Theresponse to a test dose is expressed as the fraction of the maximumactivation by 100 nM DAMGO (I_(test)/I_(max)) Data are presented asmean±SEM (n=4-5). All oocytes were used only once to avoiddesensitization.

FIG. 4: The nucleic acid sequence of the most common allele of the muopioid receptor (SEQ ID NO:1) (GENBANK accession number L25119).

FIG. 5: Amino acid sequence of the most common human mu opioid receptor(SEQ ID NO:2) referred to hMOR1, which is compared to the rat homologsof the mu (rMOR1) (SEQ ID NO:3), delta (rDOR1) (SEQ ID NO:4) and kappa(rKOR) (SEQ ID NO:5) opioid receptor amino acid sequences by the use theprogram PILEUP. Boldface type and shading, transmembrane domaincandidates; *, consensus sites for N-linked glycosylation; italics,amino acid residues different between rat and human mu opioid receptor;@, indicates intron/exon boundary.

FIGS. 6A and 6B: Inhibition of adenylyl cyclase in AV12 cells expressingeither the C17T or prototype receptor. Adenylyl cyclase in intact cellswas maximally stimulated with forskolin, and inhibition of cAMPproduction was determined in triplicate at the indicated concentrationof β-endorphin (A) or leu-enkephalin (B). Data are normalized to allowready comparison of the 1C50 values (the maximum inhibition is definedas 0 and the cyclase activity in the absence of agonist is defined as1). The actual value for maximum inhibition ranged from 77% to 95%.

DETAILED DESCRIPTION OF THE INVENTION

As explained above, the present invention is based upon Applicants'surprising and unexpected discovery of heretofore unknown singlenucleotide polymorphisms (SNPs) in the human mu opioid receptor, alongwith combinations thereof. Furthermore, Applicants have discovered thatmore than one SNP can be present in either or both alleles of the humanmu opioid receptor gene in a subject.

In addition, the present invention is based upon Applicants' surprisingdiscovery of molecules of heretofore unknown isolated nucleic acidmolecules which encode human mu opioid receptors, wherein the DNAsequences include a combination of presently known polymorphisms andsubsequently of the human mu opioid receptors discovered by Applicantsand set forth herein.

Furthermore, the present invention is based upon Applicants' surprisingand unexpected discovery that the expression of variant alleles of thehuman mu opioid gene comprising a DNA sequence having a variation in SEQID NO:1, wherein the variations comprise A118G or C17T, produce avariant mu opioid receptor comprising an amino acid sequence having avariation in SEQ ID NO:2, wherein the variations comprise Asn40Asp orAla6Val, and that these variant receptors exhibit a binding affinity forβ-endorphin that is different from the binding affinity of a mu opioidreceptor comprising an amino acid sequence of SEQ ID NO:2, and isencoded by the predominant or “most common” allele of the mu opioidreceptor gene comprising a DNA sequence of SEQ ID NO:1.

Furthermore, the present invention is based upon Applicants' predictionthat variant alleles of the mu opioid receptor gene, which comprise aDNA sequence having a variation in SEQ ID NO:1, wherein the variationcomprises A118G or C17T encode variant mu opioid receptors comprisingamino acid sequence having a variation in SEQ ID NO:2 wherein thevariation comprises Asn40Asp or Ala6Val, and the variant receptors havean activity in vivo different from the of the predominant or “mostcommon” mu opioid receptor, the presence of such variant alleles in abodily sample from a subject is expected to be indicative of theactivity of the mu opioid receptors in the subject.

The present invention further extends to heretofore unknownpolymorphisms of the human mu opioid receptor gene that can serve asgenetic markers to map the locus of the human mu opioid receptor gene.

Moreover, the present invention extends to the characterization of thebinding properties of human mu opioid receptors produced from theexpression of nucleic acid molecules comprising DNA sequences with suchheretofore unknown polymorphisms of the human mu opioid receptor gene,or combinations of heretofore unknown polymorphisms and knownpolymorphisms. In particular, the human mu opioid receptor is the majorpharmacological target for clinically important opioid alkaloids,including morphine, methadone and fentanyl, as well as for endogenousopioid peptides such as Pendorphin, Met-enkephalin-Arg-Phe, the recentlyidentified endomorphins [Zadina, J. E., Hackler, L., Ge, L. J. & Kastin,A. J. (1997) Nature 386, 499-502, which is hereby incorporated byreference in its entirety] and other opioid drugs [Pasternak, G. W.(1993) Clin. Neuropharnzacol. 16, 1-18, which is hereby incorporated byreference herein in its entirety]. Applicants have discovered that,surprisingly, human β-endorphin, an endogenous opioid, has a much higherbinding affinity for a human mu opioid receptor produced from expressionof the A118G variant allele of the human mu opioid receptor gene thanfor a human mu opioid receptor protein produced from the expression ofthe predominant or “most common” allele of the human mu opioid receptorgene (SEQ ID NO:1) comprising a DNA sequence of SEQ ID NO:1, and that avariant receptor encoded by a variant allele comprising a DNA sequenceof SEQ ID NO:1, wherein the variation comprises A118G has increasedactivity relative to the predominant or “most common” allele. Thisincreased activity is expected to result in lower activity of the HPAand HPG axes. As a result sexual and reproductive functions,gastrointestinal motility, immune response and/or ability to withstandstress are increased in the subject relative to the levels of suchfunctions in a standard comprising two alleles of the mu opioid receptorgene comprising a DNA sequence of SEQ ID NO:1.

In contrast, a human mu opioid receptor produced from expression of theC17T variant allele of the human mu opioid receptor gene has decreasedbinding affinity for β-endorphin relative to the binding affinity of ahuman mu opioid receptor protein produced from the expression of thepredominant or “most common” allele of the human mu opioid receptor gene(SEQ ID NO:1) comprising a DNA sequence of SEQ ID NO:1. Consequently, avariant receptor encoded by a C17T variant allele exhibits decreasedactivity relative to the predominant or “most common” allele. Thisdecreased activity is expected to result in increased activity of HPAand HPG axes. Hence, sexual and reproductive functions, gastrointestinalmotility, immune response and/or ability to withstand stress aredecreased in the subject relative to the levels of such physiologicalfunctions in a standard comprising two alleles of the mu opioid receptorgene comprising a DNA sequence of SEQ ID NO:1.

Furthermore, the present invention extends to characterizing theactivity of such heretofore unknown human mu opioid receptors producedfrom the expression of isolated nucleic acid molecules of the presentinvention. More particularly, the increased or decreased ability of suchhuman mu opioid receptors produced from isolated nucleic acid acids ofthe present invention to activate G protein-activated inwardlyrectifying K⁺ (GIRK) channels via a G protein-mediated mechanism can bedetermined, and is expected to be indicative of activity.

The present invention further extends to Applicants' discovery thatpolymorphisms such as A118G and C17T, are present in the population at ahigh frequency (greater than 5%), and that the presence of suchpolymorphisms in the human mu opioid receptor gene of a subjectcorrelates to an increased or decreased susceptibility to addictivediseases, such as heroin addiction, cocaine addiction, or alcoholaddiction, to name only a few, and are expected to modulatephysiological functions regulated by the HPA and HPG axes, such assexual and reproductive functions, gastrointestinal motility, immuneresponse and/or ability to withstand stress, relative to such functionsin a standard.

The present invention extends to diagnostic methods to determine asubject's increased or decreased susceptibility to at least oneaddictive disease. With the results of such methods, targeted preventionmethods, early therapeutic intervention, and improved chronic treatmentto opioid addiction are set forth herein and encompassed by the presentinvention. In addition, attending medical professionals of subjectsarmed with the results of such diagnostic methods can determine whetheradministration of opioid analgesics is appropriate or whether non-opioidderived analgesics should be administered to the subject. Also,appropriate choice and type of analgesic can be made in treating asubject's pain.

Also, the present invention extends to methods of determining asubject's increased or decreased susceptibility to pain and response toanalgesics, and using that information when prescribing analgesics tothe subject.

Furthermore, the present invention extends to diagnosing a disease ordisorder related to a physiological function regulated by the HPA andHPG axes, such as sexual and reproductive functions, gastrointestinalmotility, immune response, and the ability to withstand stress.

The present invention further extends to variant alleles of the human muopioid receptor gene comprising a DNA sequence comprising a heretoforeunknown polymorphism, such as:

G24A;

G779A; or

G942A,

or combinations thereof.

Furthermore, Applicants' invention extends to variant alleles of thehuman mu opioid receptor gene comprising a DNA sequence having at leasttwo variations in the predominant or “most common” allele comprising ahuman mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1,wherein the variations comprise

A118G;

C17T;

G24A;

G779A; or

G942A,

or a combination thereof.

Furthermore, the present invention is based on Applicants' discoverythat surprisingly and unexpectedly, the C17T variant allele of the humanmu opioid receptor is present in a statistically significantly higherfrequency in opioid dependent persons than in persons not addicted toopioids. In contrast, within an Hispanic study subject group, it wasfound that the A118G variant allele was present in a significantlyhigher proportion of non-opioid-dependent subjects compared with theopioid-dependent subjects [Yates-corrected Chisquare λ2=8.22(P=0.0041)]. This finding suggests the possibility that the A118G SNPconfers a relative protection against opioid dependence.

Consequently, an initial aspect of the present invention involvesisolation of heretofore unknown variant alleles of the human mu opioidreceptor gene. As used herein, the term “gene” refers to an assembly ofnucleotides that encode a polypeptide, and includes cDNA and genomic DNAnucleic acids.

Furthermore, in accordance with the present invention there may beemployed conventional molecular biology, microbiology, and recombinantDNA techniques within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Transkuion [B. D. Hames & S. J. Higgins, eds.(1984)); Animal Cell Culture [R. I. Freshney, ed. (1986)]; ImmobilizedCells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide ToMolecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, Inc. (1994).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” is any genetic element (e.g.,plasmnid, chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo, i.e., capable of replication under its own control.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the MnRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m) of55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5×SSC, 0.5% SDS). Moderate stringency hybhridizationconditions correspond to a higher T_(m), e.g., 40% formamide, with 5× or6×SSC. High stringency hybridization conditions correspond to thehighest T_(m), e.g., 50% formamide, 5× or 6×SSC. Hybridization requiresthat the two nucleic acids contain complementary sequences, althoughdepending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T_(m) for hybrids of nucleic acids having those sequences.The relative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (see Sambrook et al.,supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). Preferably a minimum length for ahybridizable nucleic acid is at least about 10 nucleotides; preferablyat least about 20 nucleotides; and more preferably the length is atleast about 30 nucleotides; and most preferably 40 nucleotides.

In a specific embodiment, the term “standard hybridization conditions”refers to a T_(m) of 55° C., and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 65° C.

“Homologous recombination” refers to the insertion of a foreign DNAsequence of a vector in a chromosome. Preferably, the vector targets aspecific chromosomal site for homologous recombination. For specifichomologous recombination, the vector will contain sufficiently longregions of homology to sequences of the chromosome to allowcomplementary binding and incorporation of the vector into thechromosome. Longer regions of homology, and greater degrees of sequencesimilarity, may increase the efficiency of homologous recombination.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A “promoter sequence” or “promoter” is a DNA regulatory region capableof binding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.

A coding sequence is “operatively associated with” a transcriptional andtranslational control sequences, such as a promoter for example, whenRNA polymerase transcribes the coding sequence into mRNA, which in turnis translated into a protein encoding by the coding sequence.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide, thatdirects the host cell to translocate the polypeptide. The tern“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

The phrase “expected to be indicative” is used herein to refer to thecorrelation between the identity of the allelic variation(s) in anindividual and the susceptibility of an individual to addictive disease,sensitivity to pain and analgesics, therapeutic effectiveness ofanalgesics, and other physiological manifestations described hereinrelated to the function of the mu opioid receptor, such as but notlimited to the responsiveness to stress, peripheral gastroin-testinalfunction, immune function, and reproductive biology. The correlationsare based on the findings in the present invention of the relationshipbetween the biochemistry and cellular function of the variants of the muopioid receptor and clinical observations, analyzed statistically, onhistory of drug dependence, reproductive function, gastrointestinalfunction, response to stress, and other previous or current conditions.Expected correlations of mu opioid receptor alleles and susceptibilityto various conditions may be increased susceptibility or decreasedsusceptibility.

As explained above, within the scope of the present invention are DNAsequences encoding variant alleles of a human mu opioid receptor gene ofthe present invention, which comprise at least one variation in thepredominant or “most common” allele of the human mu opioid receptorgene. The most common allele comprises a DNA sequence of SEQ ID NO:1,and variations in the most common allele comprise:

G24A;

G779A; or

G942A,

or combinations thereof.

In another embodiment, the present invention comprises DNA sequencesencoding variant alleles of a human mu opioid receptor gene, comprisingat least two variations in the predominant or “most common” allele ofthe human mu opioid receptor gene, wherein the most common human muopioid receptor gene comprises a DNA sequence of SEQ ID NO:1. Variantalleles of the human mu opioid receptor gene encompassed by the presentinvention comprise a DNA sequence comprising at least two variations ofSEQ ID NO:1, wherein the variation comprises:

G24A;

G779A;

G942A;

A118G; or

C17T.

Moreover, due to degenerate nature of codons in the genetic code,variant human mu opioid receptor proteins encoded by variant alleles ofthe present invention, wherein the variant human mu opioid receptorscomprise an amino acid sequence having at least one variation in SEQ IDNO:2, wherein the variations comprise:

Asn40Asp or conserved variants thereof;

Ala6Val or conserved variants thereof; or

Arg260His or conserved variants thereof,

or combinations thereof, can be encoded by nucleic acid molecules otherthan those set forth above. “Degenerate nature” refers to the use ofdifferent three-letter codons to specify a particular amino acidpursuant to the genetic code. It is well known in the art that thefollowing codons can be used interchangeably to code for each specificamino acid:

Phenylalanine (Phe or F) UUU or UUC

Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG

Isoleucine (Ile or I) AUU or AUC or AUA

Methionine (Met or M) AUG

Valine (Val or V) GUU or GUC of GUA or GUG

Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC

Proline (Pro or P) CCU or CCC or CCA or CCG

Threonine (Thr or T) ACU or ACC or ACA or ACG

Alanine (Ala or A) GCU or GCG or GCA or GCG

Tyrosine (Tyr or Y) UAU or UAC

Histidine (His or H) CAU or CAC

Glutamine (Gln or Q) CAA or CAG

Asparagine (Asn or N) AAU or AAC

Lysine (Lys or K) AAA or AAG

Aspartic Acid (Asp or D) GAU or GAC

Glutamic Acid (Glu or E) GAA or GAG

Cysteine (Cys or C) UGU or UGC

Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG

Glycine (Gly or G) GGU or GGC or GGA or GGG

Tryptophan (Trp or W) UGG

Termination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

As used herein, the term “sequence homology” in all its grammaticalforms refers to the relationship between proteins that possess a “commonevolutionary origin,” including proteins from superfamilies (e.g., theimmunoglobulin superfamily) and homologous proteins from differentspecies (e.g., myosin light chain, etc.) (Reeck et al., 1987, Cell50:667).

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that do not share a commonevolutionary origin (see Reeck et al., supra). However, in common usageand in the instant application, the term “homologous,” when modifiedwith an adverb such as “highly,” may refer to sequence similarity andnot a common evolutionary origin.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than30% of the amino acids are identical, or greater than about 60% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A variant allele of the human mu opioid receptor gene of the presentinvention, whether genomic DNA or cDNA, can be isolated from any source,particularly from a human cDNA or genomic library. Methods for obtainingan allele of a human mu opioid receptor gene, variants thereof, or themost common, are well known in the art, as described above (see, e.g.,Sambrook et al., 1989, supra).

Accordingly, any human cell potentially can serve as the nucleic acidsource for the molecular cloning of a variant allele of the human muopioid receptor gene of the present invention, or a nucleic acidmolecule hybridizable to a variant allele of a human mu opioid receptorgene of the present invention. The DNA may be obtained by standardprocedures known in the art from cloned DNA (e.g., a DNA “library”), andpreferably is obtained from a cDNA library prepared from tissues withhigh level expression of a human mu opioid receptor protein, by chemicalsynthesis, by cDNA cloning, or by the cloning of genomic DNA, orfragments thereof, purified from the desired cell (See, for example,Sambrook et al., 1989, supra; Glover, D. M. (ed.), 1985, DNA Cloning: APractical Approach, MRL Press, Ltd., Oxford, U.K. Vol. 1, II). Clonesderived from genomic DNA may contain regulatory and intron DNA regionsin addition to coding regions; clones derived from cDNA will not containintron sequences. Whatever the source, an allele of a human mu opioidreceptor gene of the present invention should be molecularly cloned intoa suitable vector for propagation.

In the molecular cloning of a human mu opioid receptor gene of thepresent invention, DNA fragments are generated, some of which willencode an allele. The DNA may be cleaved at specific sites using variousrestriction enzymes. Alternatively, one may use DNAse in the presence ofmanganese to fragment the DNA, or the DNA can be physically sheared, asfor example, by sonication. The linear DNA fragments can then beseparated according to size by standard techniques, including but notlimited to, agarose and polyacrylamide gel electrophoresis and columnchromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing an allele of a human mu opioid receptor of thepresent invention may be accomplished in a number of ways. For example,if an amount of a portion of an allele of a human mu opioid receptorgene, or its specific RNA, or a fragment thereof, is available and canbe purified and labeled, the generated DNA fragments may be screened bynucleic acid hybridization to the labeled probe (Benton and Davis, 1977,Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci.U.S.A. 72:3961). For example, a set of oligonucleotides corresponding tothe partial amino acid sequence information obtained for a human muopioid receptor protein can be prepared and used as probes for DNAencoding a variant allele of a human mu opioid receptor gene of thepresent invention, as was done in a specific example, infra, or asprimers for cDNA or mRNA (e.g., in combination with a poly-T primer forRT-PCR). Preferably, a fragment is selected that is highly unique to avariant allele of the human mu opioid receptor gene of the invention.Those DNA fragments with substantial homology to the probe willhybridize. As noted above, the greater the degree of homology, the morestringent hybridization conditions can be used.

Further selection can be carried out on the basis of the properties ofan allele of a human mu opioid receptor gene of the present inventione.g., if the allele encodes a variant human mu opioid receptor proteinhaving an isoelectric, electrophoretic, amino acid composition, orpartial amino acid sequence different from that produced from theexpression of the most common allele of a human mu opioid receptor gene(SEQ ID NO:1) herein. Thus, the presence of an allele of a human muopioid receptor gene of the present invention may be detected by assaysbased on the physical, chemical, or immunological properties of itsexpressed product. For example, cDNA clones, or DNA clones whichhybrid-select the proper mRNAs, can be selected which produce a proteinthat, e.g., has different electrophoretic migration, isoelectricfocusing or non-equilibrium pH gel electrophoresis behavior, proteolyticdigestion maps, or antigenic properties as known for a human mu opioidreceptor produced from expression of a most common allele of the humanmu opioid receptor gene (SEQ ID NO:1).

An allele of a human mu opioid receptor gene of the present inventioncan also be identified by mRNA selection, i.e., by nucleic acidhybridization followed by in vitro translation. In this procedure,nucleotide fragments are used to isolate complementary mRNAs byhybridization. Such DNA fragments may represent available, purified DNAof an allele of a human mu opioid receptor gene of the presentinvention, or may be synthetic oligonucleotides designed from thepartial amino acid sequence information. Immunoprecipitation analysis orfunctional assays of the in vitro translation products of the productsof the isolated mRNAs identifies the mRNA and, therefore, thecomplementary DNA fragments, that contain the desired sequences.

A labeled cDNA of an allele of a human mu opioid receptor gene of thepresent invention, or fragments thereof, or a nucleic acid hybridizableunder standard hybridization conditions to an allele of a human muopioid receptor gene of the present invention, can be synthesized usingsequences set forth herein. The radiolabeled mRNA or cDNA may then beused as a probe to identify homologous DNA fragments from among othergenomic DNA fragments. Suitable labels include enzymes, radioactiveisotopes, fluorophores (e.g., fluorescene isothiocyanate (FITC),phycoerythrin (PE), Texas red (TR), rhodamine, free or chelatedlanthanide series salts, especially Eu³⁺, to name a few fluorophores),chromophores, radioisotopes, chelating agents, dyes, colloidal gold,latex particles, ligands (e.g., biotin), and chemiluminescent agents.When a control marker is employed, the same or different labels may beused for the receptor and control marker.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re areused, known currently available counting procedures may be utilized. Inthe instance where the label is an enzyme, detection may be accomplishedby any of the presently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art. Direct labels are one example of labels which can be usedaccording to the present invention. A direct label has been defined asan entity, which in its natural state, is readily visible, either to thenaked eye, or with the aid of an optical filter and/or appliedstimulation, e.g., U.V. light to promote fluorescence. Among examples ofcolored labels, which can be used according to the present invention,include metallic sol particles, for example, gold sol particles such asthose described by Leuvering (U.S. Pat. No. 4,313,734); dye solparticles such as described by Gribnau et al. (U.S. Pat. No. 4,373,932)and May et al. (WO 88/08534); dyed latex such as described by May,supra, Snyder (EP-a 0 280 559 and 0 281 327); or dyes encapsulated inliposomes as described by Campbell et al. (U.S. Pat. No. 4,703,017).Other direct labels include a radionucleotide, a fluorescent moiety or aluminescent moiety. In addition to these direct labeling devices,indirect labels comprising enzymes can also be used according to thepresent invention. Various types of enzyme linked immunoassays are wellknown in the art, for example, alkaline phosphatase and horseradishperoxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactatedehydrogenase, urease, these and others have been discussed in detail byEva Engvall in Enzyme Inmunoassay ELISA and EMIT in Methods inEnzymology, 70. 419439, 1980 and in U.S. Pat. No. 4,857,453.

Other labels for use in the invention include magnetic beads or magneticresonance imaging labels.

Cloning Vectors

The present invention also relates to cloning vectors comprising variantalleles of a human mu opioid receptor gene of the present invention, andan origin of replication. For purposes of this Application, an “originof replication refers to those DNA sequences that participate in DNAsynthesis.

As explained above, in an embodiment of the present invention, variantalleles of a human mu opioid receptor gene of the present inventioncomprise a DNA sequence having at least one variation in the most commonallele of a human mu opioid receptor gene comprising a DNA sequence ofSEQ ID NO:1, wherein the variation comprises:

G24A;

G779A; or

G942A,

or combinations thereof.

In another embodiment, the present invention extends to variant allelesof a human mu opioid receptor gene, comprising a DNA sequence having atleast two variations in the DNA sequence of SEQ ID NO:1, wherein thevariations comprise:

G24A;

G779A;

G942A;

A118G; or

C17T.

Furthermore, an isolated variant allele of a human mu opioid receptorgene of the present invention, or isolated nucleic acid moleculeshybridizable under standard hybridization conditions to an isolatedvariant allele of a human mu opioid receptor gene of the presentinvention, can be inserted into an appropriate cloning vector in orderto produce multiple copies of the variant allele or isolated nucleicacid molecule. A large number of vector-host systems known in the artmay be used. Possible vectors include, but are not limited to, plasmidsor modified viruses. The vector system used however must be compatiblewith the host cell used. Examples of vectors include having applicationsherein, but are not limited to E. coli, bacteriophages such as lambdaderivatives, or plasmids such as pBR322 derivatives or pUC plasmidderivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc. The insertion intoa cloning vector can, for example, be accomplished by ligating a variantallele of the human mu opioid receptor gene of the present invention, oran isolated nucleic acid hybridizable thereto under standardhybridization conditions, into a cloning vector which has complementarycohesive termini. However, if the complementary restriction sites usedto fragment the variant allele or isolated nucleic acid hybridizablethereto are not present in the cloning vector, the ends of the variantallele or the isolated nucleic acid molecule hybridizable thereto understandard hybridization conditions may be enzymatically modified.Alternatively, any site desired may be produced by ligating nucleotidesequences (linkers) onto the DNA termini; these ligated linkers maycomprise specific chemically synthesized oligonucleotides encodingrestriction endonuclease recognition sequences. Such recombinantmolecules can then be introduced into host cells via transformation,transfection, infection, electroporation, etc., so that many copies of avariant allele of a human mu opioid receptor gene of the presentinvention, or an isolated nucleic acid molecule hybridizable theretounder standard hybridization conditions, can be generated. Preferably,the cloned isolated variant is contained on a shuttle vector plasmid,which provides for expansion in a cloning cell, e.g., E. coli, andfacile purification for subsequent insertion into an appropriateexpression cell line, if such is desired. For example, a shuttle vector,which is a vector that can replicate in more than one type of organism,can be prepared for replication in both E. coli and Saccharonycescerevisiae by linking sequences from an E. coli plasmid with sequencesfrom the yeast 21 plasmid.

In an alternative method an isolated variant allele of a human mu opioidreceptor gene of the present invention or an isolated nucleic acidmolecule hybridizable thereto under standard hybridization conditionsmay be identified and isolated after insertion into a suitable cloningvector in a “shot gun” approach. Enrichment for a variant allele, forexample, by size fractionation, can be done before insertion into thecloning vector.

Expression Vectors

As stated above, the present invention extends to an isolated variantallele of a human mu opioid receptor gene, comprising a DNA sequencehaving at least one variation in the DNA sequence of the predominant or“most common” allele of the human mu opioid receptor gene comprising aDNA sequence of SEQ ID NO:1 wherein the variations comprise:

G24A;

G779A; or

G942A,

or combinations thereof.

In another embodiment, the present invention extends to an isolatedvariant allele of a human mu opioid receptor gene, a DNA sequence havingat least two variations in the predominant or “most common” allele ofthe human mu opioid receptor gene comprising a DNA sequence of SEQ IDNO:1 wherein the variations comprise:

G24A;

G779A;

G942A;

A118G; or

C17T.

Variant alleles of the present invention, along with isolated nucleicacid molecules hybridizable to such variant alleles under standardhybridization conditions, can be inserted into an appropriate expressionvector, i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted protein-coding sequence.Thus, a variant allele of the present invention, or an isolated nucleicacid molecule hybridizable to a variant allele of the present inventionunder standard hybridization conditions, is operatively associated witha promoter in an expression vector of the invention. A DNA sequence is“operatively associated” to an expression control sequence, such as apromoter, when the expression control sequence controls and regulatesthe transcription and translation of that DNA sequence. The term“operatively associated” includes having an appropriate start signal(e.g., ATG) in front of the DNA sequence to be expressed and maintainingthe correct reading frame to permit expression of the DNA sequence underthe control of the expression control sequence and production of thedesired product encoded by the DNA sequence. If a variant allele of thepresent invention, or an isolated nucleic acid hybridizable theretounder standard hybridization conditions does not contain an appropriatestart signal, such a start signal can be inserted into the expressionvector in front of (5′ of) the molecule.

Both cDNA and genomic sequences can be cloned and expressed undercontrol of such regulatory sequences. An expression vector alsopreferably includes a replication origin.

The necessary transcriptional and translational signals can be providedon a recombinant expression vector, or they may be supplied by an allelecomprising a human mu opioid receptor gene.

Potential host-vector systems include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

A variant allele of a human mu opioid receptor gene of the presentinvention or an isolated nucleic acid molecule hybridizable theretounder standard hybridization conditions may be expressed chromosomally,after integration of the coding sequence by recombination. In thisregard, any of a number of amplification systems may be used to achievehigh levels of stable gene expression (See Sambrook et al., 1989,supra).

A unicellular host transformed or transfected with an expression vectorof the present invention is cultured in an appropriate cell culturemedium that provides for expression by the unicellular host of thevariant allele, or isolated nucleic acid hybridizable thereto understandard hybridization conditions.

Any of the methods previously described for the insertion of DNAfragments into a cloning vector may be used to construct expressionvectors of the present invention. These methods may include in vitrorecombinant DNA and synthetic techniques and in vivo recombination(genetic recombination).

Expression of a variant allele of a human mu opioid receptor gene of thepresent invention or an isolated nucleic acid molecule hybridizable to avariant allele of a human mu opioid receptor gene under standardhybridization conditions, may be controlled by any promoter/enhancerelement known in the art, but these regulatory elements must befunctional in the host selected for expression. Promoters which may beused to control expression include, but are not limited to, the SV40early promoter region (Benoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:3942); prokaryotic expression vectorssuch as the β-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc.Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, etal., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also “Usefulproteins from recombinant bacteria” in Scientific American, 1980,242:74-94; promoter elements from yeast or other fungi such as the Gal 4promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerolkinase) promoter, alkaline phosphatase promoter; and the animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.50:39949; MacDonald, 1987, Hepatology 7:425-515); insulin gene controlregion which is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58),alpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadal releasing hormone gene control regionwhich is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

Moreover, expression vectors comprising a variant allele of a human muopioid receptor gene of the present invention, or an isolated nucleicacid molecule hybridizable thereto under standard hybridizationconditions, can be identified by four general approaches: (a) PCRamplification of the desired plasmid DNA or specific mRNA, (b) nucleicacid hybridization, (c) presence or absence of selection marker genefunctions, and (d) expression of inserted sequences. In the firstapproach, the variant allele or isolated nucleic acid moleculehybridizable thereto under standard hybridization conditions can beamplified by PCR to provide for detection of the amplified product. Inthe second approach, the presence of a foreign gene inserted into anexpression vector of the present invention can be detected by nucleicacid hybridization using probes comprising sequences that are homologousto an inserted marker gene. In the third approach, the recombinantvector/host system can be identified and selected based upon thepresence or absence of certain “selection marker” gene functions (e.g.,β-galactosidase activity, thymidine kinase activity, resistance toantibiotics, transformation phenotype, occlusion body formation inbaculovirus, etc.) caused by the insertion of foreign genes in thevector. In yet another example, if an isolated variant allele of a humanmu opioid receptor gene of the present invention, or an isolated nucleicacid molecule hybridizable thereto under standard hybridizationconditions, is inserted within the “selection marker” gene sequence ofthe vector, recombinants containing the insert can be identified by theabsence of the inserted gene function. In the fourth approach,recombinant expression vectors can be identified by assaying for theactivity, biochemical, or immunological characteristics of the geneproduct expressed by the recombinant, provided that the expressedprotein assumes a functionally active conformation.

Naturally, the present invention extends to a method of producing avariant human mu opioid receptor comprising an amino acid sequencehaving at least one variation in the amino acid sequence of SEQ ID NO:2,wherein the variation comprises Arg260His or conserved variants thereof.An example of such a method comprises the steps of culturing aunicellular host transformed or transfected with an expression vectorcomprising a variant allele of a human mu opioid receptor genecomprising a DNA sequence having a variation in SEQ ID NO:1, wherein thevariation comprises G779A, wherein the variant allele which isoperatively associated with a promoter. The transformed or transfectedunicellular host is then cultured under conditions that provide forexpression of the variant allele of the human mu opioid receptor gene,and the expression product is recovered from the unicellular host.

Another example involves culturing a unicellular host transformed ortransfected with an isolated nucleic acid molecule hybridizable understandard hybridization conditions to a variant allele of a human muopioid receptor gene comprising a DNA sequence having at least onevariation in SEQ ID NO:1, wherein the variation comprises G779A, whereinthe isolated nucleic acid molecule is operatively associated with apromotor. The variant human mu opioid receptor is then recovered fromthe host.

In another embodiment, the present invention extends to a method forproducing a variant human mu opioid receptor comprising an amino acidsequence having at least two variations in SEQ ID NO:2, wherein thevariations comprise

Asn40Asp or conserved variants thereof;

Ala6Val or conserved variants thereof; or

Arg260His or conserved variants thereof,

such a method comprises the steps of culturing a unicellular hosttransformed or transfected with an expression vector comprising avariant allele of a human mu opioid receptor gene of the presentinvention or an isolated nucleic acid molecule hybridizable theretounder standard hybridization conditions, and operatively associated witha promoter, that provides for expression of the variant allele or theisolated nucleic acid molecule hybridizable thereto under standardhybridization conditions. After expression, a variant human mu opioidreceptor of the present invention is recovered from the unicellularhost.

A wide variety of unicellular host/expression vector combinations may beemployed in expressing the DNA sequences of this invention. Usefulexpression vectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol E1, pCR1, pBR322, pMa1-C2, pET, pGEX (Smith et al., 1988, Gene67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAS,e.g., the numerous derivatives of phage λ, e.g., NM989, and other phageDNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmidssuch as the 2μ plasmid or derivatives thereof; vectors useful ineukaryotic cells, such as vectors useful in insect or mammalian cells;vectors derived from combinations of plasmids and phage DNAs, such asplasmids that have been modified to employ phage DNA or other expressioncontrol sequences; and the like.

For example, in a baculovirus expression systems, both non-fusiontransfer vectors, such as but not limited to pVL941 (BamH1 cloning site;Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1, NotI, XmaIII, BglII, andPstI cloning site; Invitrogen), pVL1392 (BglII, PstI, NotI, XmaI, EcoRI,XbaI, SmaI, and BamH1 cloning site; Summers and Invitrogen), andpBlueBacIII (BamH1, BglII, PstI, NcoI, and HindIII cloning site, withblue/white recombinant screening possible; Invitrogen), and fusiontransfer vectors, such as but not limited to pAc700 (BamH1 and KpnIcloning site, in which the BamH1 recognition site begins with theinitiation codon; Summers), pAc701 and pAc702 (same as pAc700, withdifferent reading frames), pAc360 (BamH1 cloning site 36 base pairsdownstream of a polyhedrin initiation codon; Invitrogen(195)), andpBlueBacHisA, B, C (three different reading frames, with BamH1, Bgm,PstI, NcoI, and HindIII cloning site, an N-terminal peptide for ProBondpurification, and blue/white recombinant screening of plaques;Invitrogen (220)) can be used.

Mammalian expression vectors contemplated for use in the inventioninclude vectors with inducible promoters, such as the dihydrofolatereductase (DHFR) promoter, e.g., any expression vector with a DHFRexpression vector, or a DHFRlmethotrexate co-amplification vector, suchas pED PstI, SmaI, SbaI, SmaI, and EcoRI cloning site, with the vectorexpressing both the cloned gene and DHFR; see Kaufman, Current Protocolsin Molecular Biology, 16.12 (1991).

Alternatively, a glutamine synthetaselmethionine sulfoximineco-amplification vector, such as pEE14 (HindIII, XbaI, SmaI, SbaI,EcoRI, and BclI cloning site, in which the vector expresses glutaminesynthase and the cloned gene; Celltech). In another embodiment, a vectorthat directs episomal expression under control of Epstein Barr Virus(EBV) can be used, such as pREP4 (BamH1, SfiI, XhoI, NotI, NheI,HindIII, NheI, PvuII, and KpnI cloning site, constitutive RSV-LTRpromoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamH1, SifI,XhoI, NotI, AheI, HindIII, NheI, PvuII, and KpnI cloning site,constitutive hCMV innnediate early gene, hygromycin selectable marker;Invitrogen), pMEP4 (KpnI, PvuI, AheI, HindIII, NotI, XhoI, SfiI, BamH1cloning site, inducible methallothionein IIa gene promoter, hygromycinselectable marker: Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI,and KpnI cloning site, RSV-LTR promoter, histidinol selectable marker;Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI, and BamHIcloning site, RSV-LTR promoter, G418 selectable marker; Invitrogen), andpEBVHis (RSV-LTR promoter, hygromycin selectable marker, N-terminalpeptide purifiable via ProBond resin and cleaved by enterokinase;Invitrogen). Selectable mammalian expression vectors for use in theinvention include pRc/CMV (HindIII, BstXI, NotI, SbaI, and ApaI cloningsite, G418 selection; Invitrogen), pRc/RSV (HindIII, SpeI, BstXI, NotI,XbaI cloning site, G418 selection; Invitrogen), and others. Vacciniavirus mammalian expression vectors (see, Kaufman, 1991, supra) for useaccording to the invention include but are not limited to pSC11 (Smaicloning site, TK- and β-gal selection), pMJ601 (SalI, SmaI, AflI, NarI,BspMI, BamH1, ApaI, NheI, SaclI, KpnI, and HindIII cloning site; TK- andβ-gal selection), and pTKgptFlS (EcoRI, PstI, SalI, AccI, HindIII, SbaI,BamH1, and Hpa cloning site, TK or XPRT selection).

Yeast expression systems can also be used according to the invention toproduce a variant human mu opioid receptor or the present invention. Forexample, the non-fusion pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI,EcoRI, BstXI, BamH1, SacI, Kpn1, and HindIII cloning sit; Invitrogen) orthe fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotI, BstXI, EcoRI, BamH1,SacI, KpnI, and HindIII cloning site, N-terminal peptide purified withProBond resin and cleaved with enterokinase; Invitrogen), to mentionjust two, can be employed according to the invention.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to the following vectors or their derivatives: humanor animal viruses such as vaccinia virus or adenovirus; insect virusessuch as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

Examples of unicellular hosts contemplated by the present inventioninclude, but are not limited to E. coli Pseudonomas, Bacillus,Streptomyces, yeast, CHO, R1.1, BSW, L-M, COS1, COS7, BSC1, BSC40, BMT10and Sf9 cells. In addition, a host cell strain may be chosen whichmodulates the expression of a variant allele comprising a human muopioid receptor gene, or an isolated nucleic acid hybridizable theretounder standard hybridization conditions, such that the gene product ismodified and processed in the specific fashion desired. Different hostcells have characteristic and specific mechanisms for the translationaland post-translational processing and modification (e.g., glycosylation,cleavage [e.g., of signal sequence]) of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce an nonglycosylated coreprotein product. However, a translocation signal sequence of an isolatedvariant allele of a human mu opioid receptor gene of the presentinvention, or an isolated nucleic acid hybridizable thereto understandard hybridization conditions, expressed in bacteria may not beproperly spliced. Expression in yeast can produce a glycosylatedproduct. Expression in eukaryotic cells can increase the likelihood of“native” glycosylation and folding. Moreover, expression in mammaliancells can provide a tool for reconstituting, or constituting activity ofthe variant human mu opioid receptor gene. Furthermore, differentvector/host expression systems may affect processing reactions, such asproteolytic cleavages, to a different extent.

Vectors are introduced into the desired unicellular hosts by methodsknown in the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartnut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

An isolated variant human mu opioid receptor of the present inventionproduced as an integral membrane protein can be isolated and purified bystandard methods. Generally, the variant human mu opioid receptor can beobtained by lysing the membrane with detergents, such as but not limitedto, sodium dodecyl sulfate (SDS), Triton X-100, nonidet PA40 (NP-40),digoxin, sodium deoxycholate, and the like, including mixtures thereof.Solubilization can be enhanced by sonication of the suspension. Solubleforms of an isolated variant of a human mu opioid receptor can beobtained by collecting culture fluid, or solubilizing inclusion bodies,e.g., by treatment with detergent, and if desired sonication or othermechanical processes, as described above. The solubilized or solubleprotein can be isolated using various techniques, such as polyacrylamidegel electrophoresis (PAGE), isoelectric focusing, 2-dimensional gelelectrophoresis, chromatography (e.g., ion exchange, affmity,immunoaffmity, and sizing column chromatography), centrifugation,differential solubility, immunoprecipitation, or by any other standardtechnique for the purification of proteins.

Due to the degeneracy of nucleotide coding sequences, other DNAsequences which encode the variant human mu opioid receptors of thepresent invention may be used in the practice of the present invention.These include but are not limited to allelic genes, homologous genesfrom other species, and nucleotide sequences comprising all or portionsof genes which are altered by the substitution of different codons thatencode the same amino acid residue within the sequence, thus producing asilent change. Likewise, the conserved variants of human mu opioidreceptors of the present invention include, but are not limited to,those containing, as a primary amino acid sequence, substitutions ofamino acids in a variant human mu opioid receptor as set forth above,which are functionally equivalent to amino acids of the variations setforth above, resulting in a conservative amino acid substitution. Forexample, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity, which acts as afunctional equivalent, resulting in a silent alteration. Substitutes foran amino acid within the sequence may be selected from other members ofthe class to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. Amino acidscontaining aromatic ring structures are phenylalanine, tryptophan, andtyrosine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations will not be expected to affect apparentmolecular weight as determined by polyacrylamide gel electrophoresis, orisoelectric point.

Particularly preferred substitutions are:

Lys for Arg and vice versa such that a positive charge may bemaintained;

Glu for Asp and vice versa such that a negative charge may bemaintained;

Ser for Thr such that a free —OH can be maintained; and

Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced at a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

Antibodies to Variant Human mu Opioid Receptors of the Present Invention

According to the invention, variant human mu opioid receptors disclosedherein may be used as an immunogen to generate antibodies that recognizethe claimed variant mu opioid receptors. Such antibodies include but arenot limited to polyclonal, monoclonal, chimeric, single chain, Fabfragments, and an Fab expression library. Furthermore, antibodies of theinvention may be cross reactive, e.g., they may recognize human muopioid receptors comprising an amino acid sequence of SEQ ID NO:1, aswell as mu opioid receptors from different species. Polyclonalantibodies have greater likelihood of cross reactivity. Alternatively,an antibody of the invention may be specific for a specific variantallele of a mu opioid receptor.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to variant opioid receptors disclosed herein. Forthe production of an antibody, various host animals can be immunized byinjection with a variant human mu opioid receptor of the invention,including but not limited to rabbits, mice, rats, sheep, goats, etc. Inone embodiment, the variant human mu opioid receptor can be conjugatedto an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyholelimpet hemocyanin (KLH). Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a particularhuman mu opioid receptor of the present invention, any technique thatprovides for the production of antibody molecules by continuous celllines in culture may be used. These include but are not limited to thehybridoma technique originally developed by Kohler and Milstein [Nature256:495-497 (1975)], as well as the trioma technique, the human B-cellhybridoma technique [Kozbor et al., Immunology Today 4:72 1983); Cote etal., Proc. Nati. Acad. Sci. U.S.A. 80:202-2030 (1983)], and theEBV-hybridoma technique to produce human monoclonal antibodies [Cole etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96 (1985)]. In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizingrecent technology [PCT/US90/02545]. In fact, according to the invention,techniques developed for the production of “chimeric antibodies”Morrison et al., J. Bacteriol. 159:870 (1984); Neuberger et al., Nature312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)] bysplicing the genes from a mouse antibody molecule specific for a varianthuman mu opioid receptor of the present invention together with genesfrom a human antibody molecule of appropriate biological activity can beused; such antibodies are within the scope of this invention. Such humanor humanized chimeric antibodies are preferred for use in determiningthe presence of a particular human mu opioid receptor in a sample takenfrom a subject.

According to the invention, techniques described for the production ofsingle chain antibodies [U.S. Pat. Nos. 5,476,786 and 5,132,405 toHuston; U.S. Pat. No.4,946,778] can be adapted to produce particularvariant mu opioid receptor-specific single chain antibodies. Anadditional embodiment of the invention utilizes the techniques describedfor the construction of Fab expression libraries [Huse et al., Science246:1275-1281(1989)] to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for a variant muopioid receptor of the present invention.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of a variant human mu opioid receptor of the presentinvention, one may assay generated hybridomas for a product which bindsto a fragment of the variant human mu opioid receptor containing suchepitope.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of a variant human mu opioidreceptor, e.g., for Western blotting, imaging a variant human mu opioidreceptor in situ, measuring levels thereof in appropriate physiologicalsamples, etc. using any of the detection techniques mentioned above orknown in the art.

Yet another embodiment is based on Applicants' remarkable discovery thatsurprisingly, certain variant human mu opioid receptors have greateraffinity for particular opioids than human mu opioid receptorscomprising an amino acid sequence of SEQ ID NO:2. More specifically,Applicants have discovered that surprisingly, a variant human mu opioidreceptor produced from expression of a variant allele of a human muopioid receptor comprising a variation in SEQ ID NO:1, wherein thevariation comprises A118G, binds three times more tightly toβ-endorphin, an endogenous opioid comprising 31 amino acid residues,than do human mu opioid receptors produced from the expression of thepredominant or “most common” allele comprising a DNA sequence of SEQ IDNO:1. Since β-endorphin is believed to play an important role innumerous physiological functions, the presence of a variant comprisingA118G, in either or both alleles present in a subject has an impact onsuch physiological functions. Furthermore, β-endorphin induced activityof a receptor produced from the expression of a variant allele of ahuman mu opioid receptor gene comprising the A118G polymorphism effectedthe receptor's activation of GIRK channels via a G protein-mediatedmechanism relative to the activity of a receptor produced from theexpression of the predominant or “most common” allele. Consequently, asubject having an A118G variation, in either or both alleles of a humanmu opioid receptor gene is expected to have lower susceptibility andgreater tolerance to pain relative to a person comprising two copies ofthe predominant or “most common” allele.

One such function involves a susceptibility to at least one addictivedisease, such as opioid addiction, cocaine addiction or addiction toother psychostimulants, nicotine addiction, barbiturate or sedativehypnotic addiction, anxiolytic addiction, or alcohol addition. Inparticular, Applicants have discovered a variant allele of a human muopioid receptor gene comprising a DNA sequence having at variation inSEQ ID NO:1, wherein the variation comprises A118G, is present in anHispanic study population at a statistically significant greaterfrequency in the genomes of persons not suffering from at least oneaddictive disease, relative to its presence in the genomes of personssuffering from an addictive disease, such as opioid addiction.

Moreover, the inventors herein have further discovered that anothervariant allele of a human mu opioid receptor gene comprising a DNAsequence having a variation in SEQ ID NO:1, wherein the variationcomprises C17T, is present at a statistically significant greaterfrequency in the genomes of persons suffering from at least oneaddictive disease, relative to its presence in the genome of persons notsuffering from at least one addictive disease. Furthermore, and as shownin the Examples below, it has been found that the C17T variant mu opioidreceptor has a β-endorphin binding affinity approximately 0.72 timesthat of the human mu opioid receptor produced from expression of thepredominant or “most common” allele of the human mu opioid receptorgene. The decreased binding affinity of this variant is in contrast tothe increased affinity of the A118G variant mentioned above. Inaddition, one of the important indices of cellular functioning of the muopioid receptor is the inhibition of adenylyl cyclase activity followingagonist binding to the receptor. The C17T variant mu opioid receptor hasbeen found to be less sensitive to two endogenous opioids, β-endorphinand leu-enkephalin, than the receptor produced by the “most common”allele: the EC50 values differed by a factor of approximately 2.7 and2.8, respectively.

Hence, a variant allele of a human mu opioid receptor gene comprising aDNA sequence having an A118G variation, in SEQ ID NO:1, has increasedaffinity for β-endorphin and capability of effecting activation of GIRKchannels via a G protein-mediated mechanism provides resistance againstsusceptibility to addictive diseases, while a variant allele comprisinga human mu opioid receptor having a C17T variation in SEQ ID NO:1, has adecreased affmity for β-endorphin and a decreased sensitivity toadenylyl cyclase inhibition by the endogenous ligands β-endorphin andleu-enkephalin, and is indicative of increased susceptibility to atleast one addictive disease. These differences in variant mu opioidreceptor function at the biochemical and cellular level and theircorrelations with physiological manifestations in susceptibility toaddictive disease provides a basis for the value of the identity ofalleles in identifying individual susceptibility to addictive disease,pain, reaction to therapeutic agents, etc.

Consequently, the present invention extends to a method for determininga susceptibility of a subject to one addictive disease comprisingremoving a bodily sample comprising a first and second allele of a humanmu opioid receptor gene from the subject, and determining whether eitherthe first or second alleles, or both alleles comprise a DNA sequencehaving at least one variation in SEQ ID NO:1, wherein the variationcomprises:

A118G; or

C17T.

The presence of the variation of C17T in either or both alleles of ahuman mu opioid receptor gene of a sample from the subject indicates thesubject has an increased susceptibility to at least one addictivedisease relative to a standard having alleles of the human mu opioidreceptor gene comprising a DNA sequence of SEQ ID NO:1.

Furthermore, the presence of the variation of comprising A118G, ineither or both alleles of a mu opioid receptor gene in a sample from thesubject is expected to indicate the subject as a decreasedsusceptibility to addictive diseases relative to a standard comprisingalleles of a human mu opioid receptor gene comprising a DNA sequence ofSEQ ID NO:1.

In this embodiment, the biological sample can be a biological fluid,such as but not limited to, blood, serum, plasma, interstitial fluid,plural effisions, urine, cerebrospinal fluid, and the like. Preferably,variant alleles of a human mu opioid receptor gene, as described above,are detected in serum or urine, which are both readily obtained.Alternatively, variant alleles of a human mu opioid receptor geneindicating increased or decrease susceptibility to addictive diseases inthe subject as described above, can be detected from cellular sources,such as, but not limited to, brain tissue biopsies, adipocytes, testes,heart, and the like. For example, cells can be obtained from anindividual by biopsy and lysed, e.g., by freeze-thaw cycling, ortreatment with a mild cytolytic detergent such as, but not limited to,TRITON X-100®, digitonin, NONIDET P (NP)-40®, saponin, and the like, orcombinations thereof (see, e.g., International Patent Publication WO92/08981, published May 29, 1992). In yet another embodiment, samplescontaining both cells and body fluids can be used (see ibid.).

Other methods presently understood by a skilled artisan, and encompassedby the present invention, can also be used to detect the presence ofeither variation in either or both alleles of a human mu opioid receptorgene in a sample, and hence increased or decreased susceptibility to atleast one addictive disease of the subject relative to thesusceptibility of at least one addictive disease in a standardcomprising alleles of the human mu opioid receptor gene comprising a DNAsequence of SEQ ID NO:1.

1. For example, an optionally detectably labeled isolated nucleic acidmolecule hybridizable under standard hybridization conditions to anisolated variant allele of a human mu opioid receptor gene comprising aDNA sequence having a variation in SEQ ID NO:1, wherein the variationcomprises C17T, can be used in standard Northern hybridization analysisto detect the presence, and in some instances quantitate the level oftranscription of such a variant allele of the present invention. Thepresence of this variant allele in a bodily sample from a subject isexpected to be indicative of increased susceptibility to at least oneaddictive disease in the subject. Likewise, an optionally detectablylabeled isolated nucleic acid molecule hybridizable under standardhybridization conditions to an allele of a human mu opioid receptor genecomprising a DNA sequence having a variation in SEQ ID NO:1, wherein thevariation comprises A118G can be used in a standard Northernhybridization analysis to detect the presence of a variant allele in thesample comprising a variation in SEQ ID NO:1, wherein the variationcomprises A118G, which is expected to be indicative of a decreasedsusceptibility to at least one addictive disease relative to thesusceptibility of a standard comprising two alleles of a human mu opioidreceptor gene comprising a DNA sequence of SEQ ID NO:1.

Alternatively, oligonucleotides of the invention can be used as PCRprimers to amplify an allele of a human mu opioid receptor gene of thebiological sample e.g., by reverse transcriptase-PCR, or amplificationof the allele itself. The amplified mRNA or DNA can then be quantifiedor sequenced in order to determine the presence of a variant allele, andthe susceptibility of the subject to addictive diseases. Furthermore,variations in SEQ ID NO:1, as described above, can be found by creationor deletion of restriction fragment length polymorphisms (RFLPs) notfound in the predominant or “most common” allele, hybridization with aspecific probe engineered to hybridize to variation described aboveunder standard hybridization conditions, (or lack of hybridization witha probe specific for the predominant or “most common” allele), as wellas by other techniques.

Furthermore, biochemical or immunochemical/biochemical (e.g.,immunoprecipitation) techniques can be used to detect the presence andor level of expression of a variant allele of a human mu opioid receptorgene comprising a DNA sequence having a variation in SEQ ID NO:1,wherein the variation comprises:

A118G; or

C17T.

The presence of the A118G variation in either or both alleles of a humanmu opioid receptor gene in a biological sample from the subjectindicates a decreased susceptibility to addictive diseases in thesubject, and the presence of the C17T variation in either or bothalleles of a human mu opioid receptor gene in a biological sample fromthe subject indicates increased susceptibility to addictive diseases inthe subject. For example, methods such as radioimmunoassay, ELISA(enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,inmmunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and nimunoelectrophoresisassays, etc using antibodies of the present invention, can be used todetermine the presence of a variant in an allele of a human mu opioidreceptor gene in a sample taken from the subject, and hence, thesubject's susceptibility to addictive diseases relative to thesusceptibility of a standard. In one embodiment, antibody binding isdetected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

Determining Susceptibility to Pain a Subject

In yet another embodiment, the present invention extends to a method fordetermining a susceptibility to pain in a subject. As explained above,Applicants have discovered that endogenous opioid, such as β-endorphin,bind about three times as tightly to a variant human mu opioid receptorcomprising an amino acid sequence having a in SEQ ID NO:2 wherein thevariation comprises Asn40Asp, relative to the binding of β-endorphin toa human mu opioid receptor comprising an amino acid sequence of SEQ IDNO:2, encoded by the predominant or “most common” allele of a human muopioid receptor gene comprising a DNA sequence of SEQ ID NO:1. Also,β-endorphin induced activity of a receptor produced from the expressionof a variant allele of a human mu opioid receptor gene or an isolatednucleic acid molecule hybridizable thereto under standard hybridizationconditions comprising the A118G polymorphism effected the receptor'sactivation of GIRK channels via a G protein-mediated mechanism relativeto the activity of a receptor produced from the expression of thepredominant or “most common” allele. Consequently, a subject having anA118G variation, in either or both alleles of a human mu opioid receptorgene is expected to have lower susceptibility and greater tolerance topain relative to a person comprising two copies of the predominant or“most common” allele.

Applicants have further discovered a variant allele of a human mu opioidreceptor gene comprising a DNA sequence having a variation in SEQ IDNO:1, wherein the variation comprises C17T, is present at astatistically significantly greater frequency in the genomes of opioidaddicts relative to its presence in the genomes of persons not addictedto opioids. Hence, the presence of a variant allele of a human mu opioidreceptor comprising a DNA sequence having a variation in SEQ ID NO:1,wherein the variation comprises C17T, in a biological sample taken froma subject, indicates the subject is predicted to have increasedsusceptibility and decreased tolerance to pain.

Hence, disclosed herein is a method of determining susceptibility ofpain in a subject, comprising the steps of removing a bodily samplecomprising a first and second allele of a human mu opioid receptor genefrom the subject, and determining whether either the first or secondalleles, or both alleles, comprise a DNA sequence having at least onevariation in SEQ ID NO:1, wherein the variation comprises:

A118G; or

C17T.

The presence of at least one variation in either or both alleles of thehuman mu opioid receptor gene is expected to be indicative of thesubject's increased or decreased susceptibility to pain relative to aperson homozygous with respect to the predominant or “most common”allele comprising a human mu opioid receptor gene comprising a DNAsequence of SEQ ID NO:1.

Numerous methods presently available, and understood by the skilledartisan, can be used to “genotype” a subject in regards to the presenceof a variant allele of a human mu opioid receptor gene in the genome ofthe subject. In particular, methods described above to ascertainincreased or decreased susceptibility to addictive diseases haverelevance in this embodiment of the present invention, and can readilybe used herein. For example, Northern blot hybridization an isolatednucleic acid of the present invention hybridizable under standardhybridization conditions to an isolated variant allele of a human muopioid receptor gene comprising a DNA sequence having a variation of SEQID NO:1, wherein the variation comprises:

A118G; or

C17T,

as a probe, along with RT-PCR, PCR, and numerous immunoassays describedabove, have applications herein.

Moreover, once susceptibility to pain in a subject has been determined,it is possible for attending medical professionals treating the subjectfor pain to administer an appropriate amount of pain reliever to thesubject in order to induce analgesia. More specifically, aninappropriate amount of pain reliever is administered to a subject wheneither the subject is not relieved of pain, or the subject is exposed topotential deleterious side effects of the pain reliever, such asinduction of addiction to the pain reliever, brain damage, or death.

However, since the amount of pain reliever administered to a subject ispresently based principally on weight, information regarding thegenotype of the subject with respect to the human mu opioid receptorgene can help increase accuracy in determining a therapeuticallyeffective amount of pain reliever to administer in order to induceanalgesia, making the use of pain relievers much safer for the subject.

Similarly, once ascertained, a susceptibility to addiction and responseto human mu opioid receptor directed therapeutic agents, appropriatemedications and dosages thereof can be determined for treatment ofaddictive diseases.

Diagnosing and Treating a Disease or Disorder Related to a PhysiologicalFunction Regulated by the HPA or HPG Axes

In yet another embodiment, the present invention extends to a method fordiagnosing a disease or disorder related to a physiological functionregulated by the HPA or HPG axes. Examples of such physiologicalfunctions include sexual or reproductive functions, gastrointestinalmotility, immune response, or ability to withstand stress. Moreover,examples of diseases or disorders which can be diagnosed with thepresent invention include infertility, constipation, diarrhea, anddecreased immune response to name only a few.

As explained above, Applicants have discovered that endogenous opioid,such as β-endorphin, bind about three times as tightly to a varianthuman mu opioid receptor comprising an amino acid sequence having a inSEQ ID NO:2 wherein the variation comprises Asn40Asp, relative to thebinding of β-endorphin to a human mu opioid receptor comprising an aminoacid sequence of SEQ ID NO:2, encoded by the predominant or “mostcommon” allele of a human mu opioid receptor gene comprising a DNAsequence of SEQ ID NO:1. Also, β-endorphin induced activity of areceptor produced from the expression of a variant allele of a human muopioid receptor gene or an isolated nucleic acid molecule hybridizablethereto under standard hybridization conditions comprising the A118Gpolymorphism effected the receptor's activation of GIRK channels via a Gprotein-mediated mechanism relative to the activity of a receptorproduced from the expression of the predominant or “most common” allele.Consequently, a subject having an A118G variation, in either or bothalleles of a human mu opioid receptor gene is expected to have hasincreased activity of the receptor relative to the activity of areceptor produced from expression of the predominant or “most common”allele comprising a DNA sequence of SEQ ID NO:1. This increased activityis expected to result in lower activity of the HPA and HPG axes. As aresult sexual and reproductive functions, gastrointestinal motility,immune response and/or ability to withstand stress are expected to beincreased in the subject relative to the levels of such functions in astandard comprising two alleles of the mu opioid receptor genecomprising a DNA sequence of SEQ ID NO:1.

In contrast, Applicants have discovered a human mu opioid receptorproduced from expression of the C17T variant allele of the human muopioid receptor gene is expected to have decreased binding affinity forβ-endorphin relative to the binding affinity of a human mu opioidreceptor protein produced from the expression of the predominant or“most common” allele of the human mu opioid receptor gene (SEQ ID NO:1)comprising a DNA sequence of SEQ ID NO:1. Consequently, a variantreceptor encoded by a C17T variant allele exhibits decreased activityrelative to the predominant or “most common” allele. This decreasedactivity is expected to result in increased activity of HPA and HPGaxes. Hence, sexual and reproductive functions, gastrointestinalmotility, immune response and/or ability to withstand stress areexpected to be decreased in the subject relative to the levels of suchphysiological functions in a standard comprising two alleles of the muopioid receptor gene comprising a DNA sequence of SEQ ID NO:1.

Hence, disclosed herein is a method of diagnosing a disease or disorderrelated to a physiological function regulated by the HPA or HPG axes ina subject, comprising the steps of removing a bodily sample comprising afirst and second allele of a human mu opioid receptor gene from thesubject, and determining whether either the first or second alleles, orboth alleles, comprise a DNA sequence having at least one variation inSEQ ID NO:1, wherein the variation comprises:

A118G; or

C17T.

The presence of at least one variation in either or both alleles of thehuman mu opioid receptor gene is expected to be indicative of a diseaseor disorder related to a physiological function regulated by the HPA orHPG axes relative to such functions in a person homozygous with respectto the predominant or “most common” allele comprising a human mu opioidreceptor gene comprising a DNA sequence of SEQ ID NO:1. Examples of suchphysiological functions include sexual or reproductive functions,gastrointestinal motility, immune response, or ability to withstandstress. Moreover, examples of diseases or disorders which can bediagnosed with the present invention include infertility, constipation,diarrhea, and decreased immune response to name only a few relative to aperson homozygous with respect to the predominant or “most common”allele comprising a human mu opioid receptor gene comprising a DNAsequence of SEQ ID NO:1.

Numerous methods presently available, and understood by the skilledartisan, can be used to “genotype” a subject in regards to the presenceof a variant allele of a human mu opioid receptor gene in the genome ofthe subject. In particular, methods described above to ascertainincreased or decreased susceptibility to addictive diseases haverelevance in this embodiment of the present invention, and can readilybe used herein. For example, Northern blot hybridization an isolatednucleic acid of the present invention hybridizable under standardhybridization conditions to an isolated variant allele of a human muopioid receptor gene comprising a DNA sequence having a variation of SEQID NO:1, wherein the variation comprises:

A118G; or

C17T,

as a probe, along with RT-PCR, PCR, and numerous immunoassays describedabove, have applications herein

In an alternative, such a method comprises removing a bodily sample fromthe subject comprising a mu opioid receptor, and determining whether thereceptor comprises an amino acid sequence having a variation in SEQ IDNO:1, wherein the variation comprises:

Asn40Asp or conserved variants thereof; or

Ala6Val or conserved variants thereof,

such that the presence of at least one variation is expected to beindicative of a disease or disorder related to a physiological functionregulated by the HPA or HPG axes, such as sexual function ordevelopment, gastric motility, immune response, or the ability of thesubject to withstand stress, relative to regulation of such activitiesin a standard comprises a human mu opioid receptor having an amino acidsequence of SEQ ID NO:2.

In particular, the presence of a variant human mu opioid receptorcomprising an amino acid sequence having at least one variation in SEQID NO:2 wherein the variation comprises Asn40Asp or conserved variantsthereof, is expected to be indicative of increased sexual orreproductive functions, increased gastrointestinal motility, increasedimmune response, or increased ability to withstand stress relative tothe levels of such function observed in a standard having a mu opioidreceptor comprising an amino acid sequence of SEQ ID NO:2.

Furthermore, the presence of a variant human mu opioid receptorcomprising an amino acid sequence having a variation in SEQ ID NO:2,wherein the variation comprises Ala6Val or conserved variants thereof,in a bodily sample taken from a subject is expected to be indicative ofdecreased sexual or reproductive functions, decreased gastrointestinalmotility, decreased immune response, or decreased ability to withstandstress relative to the levels of such function observed in a standardhaving a mu opioid receptor comprising an amino acid sequence of SEQ IDNO:2. Examples of specific diseases or disorders related to regulationof physiological functions regulated by the HPA or HPG axes includeinfertility, constipation, diarrhea, decreased immune response toantigens, or a lack of ability to withstand stress.

Numerous methods of detecting a variant mu opioid receptor as describedabove are presently available to the skilled artisan. For example areceptor in the bodily sample can be digested into fragments withproteases or CNBr. These fragments can then be collected and sequencedusing presently known methods. Once the sequence of the receptor hasbeen determined, it is a simple matter of comparing it to the amino acidsequence of the predominant or “most common” receptor having an aminoacid sequence of SEQ ID NO:2, to determine whether a variation in theamino acid sequence exists. Other methods involve immune assaysdescribed herein using antibodies of the present invention, or a bindingassay to determine the binding affinity of the receptor to β-endorphin.If its binding to β-endorphin is approximately 3 times greater than theknown binding affinity of the predominant or “most common” receptor forβ-endorphin, then the receptor is expected to have an amino acidsequence having a variation in SEQ ID NO:2, wherein the variationcomprises Asn40Asp or conserved variants thereof, and is expected to beindicative of a disease or disorder related to increased sexual orreproductive functions, increased gastrointestinal motility, increasedimmune response, or increased ability to withstand stress.

Moreover, once a disease or disorder related to a physiologicalcondition regulated by the HPA or HPG axes has been diagnosed, it ispossible for attending medical professionals treating the suspect toselect an appropriate therapeutic agent for treating such a disease anddisorder, and a therapeutically effective amount of such pain relieverto administer to the subject. Hence naturally, the present inventionextends to a method for selecting an appropriate therapeutic agent fortreating a disease or disorder related to a physiological functionregulated by the HPA and HPG axes, wherein such physiological functionsinclude sexual and reproductive functions, gastrointestinal motility,immune response, and ability to withstand stress. Furthermore, diseasesor disorders related to such functions which can be diagnosed with thepresent invention include, but are not limited to, infertility,constipation, diarrhea, and decreased immune response, to name only afew.

Commercial Kits

Furthermore, as explained above, the present invention extends tocommercial kits having applications in screening a bodily sample takenfrom a subject for the presence of a variant allele comprising a humanmu opioid receptor comprising a DNA sequence having a variation in SEQID NO:1, wherein the variation comprises:

A118G;

C17T.

With information obtained from the use of a test kit of the presentinvention, an attending health profession can determine whether thesubject has an susceptibility to pain relative to a standard, anincreased susceptibility to at least one addictive disease relative tothe susceptibility of a standard, a therapeutically effective amount ofpain reliever to administer to the subject suffering from pain in orderto induce analgesia in the subject relative to the therapeuticallyeffective amount of pain reliever to administer to a standard in orderto induce analgesia in the standard, or a therapeutically effectiveamount therapeutic agent to administer to a subject suffering from atleast one addictive disease, relative to the therapeutically effectiveamount of therapeutic agent to administer to standard suffering from atleast one addictive disease. Furthermore, such information can also beused to diagnose a disease or disorder related to a physiologicalfunction regulated by the HPA or HPG axes, such as sexual orreproductive functions, gastrointestinal motility, immune response, orability to withstand stress, or selecting an appropriate therapeuticagent and a therapeutically effective amount of such an agent toadminister to a subject suffering from a disease or disorder related toa physiological function regulated by the HPA or HPG axes. In each usedescribed above, the standard comprises a first and or second allele ofa human mu opioid receptor gene comprising a DNA sequence of SEQ IDNO:1.

Accordingly, a test kit of the present invention for determining whethera subject comprises a variant allele of a human mu opioid receptor genecomprising a DNA sequence having a variation in SEQ ID NO:1, comprisesmeans for detecting the presence of a variation in a first and or secondallele comprising a human mu opioid receptor in a biological sample froma subject, and optimally packaged with directions for use of the kit. Inone particular aspect, the means for detecting the presence of a variantallele of a human mu opioid receptor gene comprising a DNA sequencehaving a variation in SEQ ID NO:1, comprises a specific binding partnerof a human mu opioid receptor, such as an antibody, and means fordetecting the level of binding of the specific binding partner of theantibody to the particular human mu opioid receptor. In anotherembodiment, a test kit comprises an oligonucleotide probe for binding toa variant allele of a human mu opioid receptor gene comprising a DNAsequence having a variation in SEQ ID NO:1; and means for detecting thelevel of binding of the probe to the variant allele, wherein detectionbinding of the probe to the variant allele indicates the presence of avariant comprising a human mu opioid receptor gene comprising a DNAsequence having a variation in SEQ ID NO:1, wherein the variationcomprises:

A118G; or

C17T.

The sequence of the oligonucleotide probe used in a commercial kit willdetermine which if any variation is present in an allele comprising ahuman mu opioid receptor gene. Should no binding be detected, it isprobable that no such variation exists in either allele of the subject.

More specifically, a commercial test kit of the present inventioncomprises:

a) PCR oligonucleotide primers suitable for detection of a variantallele of a human mu opioid receptor gene comprising a DNA sequencehaving a variation in SEQ ID NO:1, as set forth above,

b) other reagents; and

c) directions for use of the kit.

Examples of PCR oligonucleotide primer suitable for detection of anallele comprising a human mu opioid receptor gene comprising a DNAsequence having a variation in SEQ ID NO:1 can be readily produced by aperson of ordinary skill in the art with teaching set forth herein, andvariations of SEQ ID NO:1 also set forth herein.

The present invention further extends to commercial test kits capable ofdetecting a variant human mu opioid receptor in a bodily sample takenfrom a subject. Examples of variant human mu opioid receptors that canbe detected with a kit of the present invention comprise:

a variant human mu opioid receptor comprising an amino acid sequencehaving a variation in SEQ ID NO:2, wherein the variation comprises thevariation comprises Arg260His or conserved variants thereof, or

a variant human mu opioid receptor comprising an amino acid sequencehaving at least two variations in SEQ ID NO:2, wherein the variationscomprise:

Asn40Asp or conserved variants thereof;

Ala6Val or conserved variants thereof; or

Arg260His or conserved variants thereof.

Moreover, a commercial test kit of the present invention can be used todetermine: a susceptibility to pain in a subject relative to a standard,an increased susceptibility to at least one addictive disease in asubject relative to the susceptibility of a standard, a therapeuticallyeffective amount of pain reliever to administer to the subject sufferingfrom pain in order to induce analgesia in the subject relative to thetherapeutically effective amount of pain reliever to administer to astandard in order to induce analgesia in the standard, a therapeuticallyeffective amount of a therapeutic agent to administer to a subjectsuffering from at least one addictive disease, relative to thetherapeutically effective amount of therapeutic agent to administer tostandard suffering from at least one addictive disease, a diagnosis of adisease or disorder related to a physiological function regulated by theHPA or HPG axes, such as sexual or reproductive functions,gastrointestinal motility, immune response, or ability to withstandstress, or selecting an appropriate therapeutic agent and atherapeutically effective amount of such an agent to administer to asubject suffering from a disease or disorder related to a physiologicalfunction regulated by the HPA or HPG axes. In each use described above,the standard comprises a first and or second allele of a human mu opioidreceptor gene comprising a DNA sequence of SEQ ID NO:1.

Accordingly, the present invention extends to a commercial test kithaving applications set forth above, comprising a predetermined amountof at least one detectably labeled immunochemically reactive componenthaving affinity for a variant human mu opioid receptor;

(b) other reagents; and

(c) directions for use of the kit.

Antibodies of the present invention, and set forth above, have readilyapplications in a commercial test kit of the present invention.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. “competitive,” “sandwich,” “double antibody,” etc.), andcomprises:

(a) a labeled component which has been obtained by coupling the human muopioid receptor of a bodily sample to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

(i) a ligand capable of binding with the labeled component (a);

(ii) a ligand capable of binding with a binding partner of the labeledcomponent (a);

(iii) a ligand capable of binding with at least one of the component(s)to be determined; and

(iv) a ligand capable of binding with at least one of the bindingpartners of at least one of the component(s) to be determined; and

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between the human mu opioid receptor gene of the presentinvention and a specific binding partner thereto.

The present invention may be better understood by reference to thefollowing non-limiting Example, which is provided as exemplary of theinvention. The following Example is presented in order to more fullyillustrate the preferred embodiments of the invention. It should in noway be construed, however, as limiting the broad scope of the invention.

EXAMPLE

The mu opioid receptor is the major target for clinically importantopioid alkaloids including morphine, methadone, fentanyl, and otheropioid drugs (1,3), as well as for endogenous opioid peptides suchβ-endorphin, Met-enkephalin-Arg-Phe, and the recently identifiedendomorphins (5). Furthermore, it is the major molecular site of actionfor heroin (2,6). Rapid activation of the mu opioid receptor, such asoccurs in the setting of drug abuse, results in a euphoric effect, thusconferring the reinforcing or rewarding effects of the drug,contributing to the development of addiction. Clinical observations havesuggested that individuals have varied sensitivity to opioids,suggesting potential variability in the receptor protein and gene.

Molecular cloning of the mu opioid receptor (7-9) has made it possibleto determine potential sequence polymorphism, as shown by a recent study(10). To further identify SNPs of the mu opioid receptor, a PCR-basedstrategy was used to amplify the coding regions of the mu opioidreceptor gene, and to determine the DNA sequence of the amplified exons.Using this method DNA samples were sequenced from 152 subjects includingboth former heroin addicts in methadone maintenance treatment andindividuals with no history of opiate or non-opiate drug dependence.

RESULTS AND DISCUSSION

Recent studies have shown that up to 90% of persons entering treatmentfor heroin addiction also abuse cocaine, alcohol or other drugs.However, following stabilization on methadone maintenancepharmacotherapy (treatment for one year or more), the percentage ofpatients who continue to abuse cocaine drops to approximately 30% andalcohol to 25 to 30% (11-13). Rigorous characterization of substanceabuse profiles was therefore obtained for all study subjects. Selfreported drug abuse history was confirmed for addictive disease patientsby routine urine toxicology. Also, since 20% to 50% of persons withchemical dependency may have a definable mental disorder which can becharacterized by standard diagnostic criteria and also since a similarlyhigh percentage of persons with a diagnosed mental illness have chemicaldependency, extensive psychological and psychiatric evaluations of studysubjects were performed (14,15).

Inclusion criteria were met by 152 individuals (refer to Methods fordetails of inclusion/exclusion criteria). One hundred thirteen of thestudy subjects (74.3%) were opiate dependent with or without previous orcurrent co-dependency for other substances; 39 study subjects (25.7%)had no history of drug dependence. Study subjects were well balancedbetween females and males: 69 females (45.4%) and 83 males (56.4%) wereincluded in the study subject pool. The ethnic breakdown of the studysubject populations was as follows: African-American 31, (20.3%),Caucasian, 52 (34.2%), Hispanic, 67 (44.1%), Native North-American, 1,(0.7%) and Other, 1 (0.7%). Although several individuals could beclassified into two separate groups if one parent came from one ethnicgroup and the other parent from another group, including fourindividuals (2.6%) who reported one parent African-American and oneCaucasian, and five individuals (3.3%) who reported one parent Caucasianand one Hispanic, for the genotype calculations the former wereclassified as African-American and the latter as Hispanic. Within thegroup of former heroin addicts in methadone maintenance treatment, themean years in treatment was 6.7 with a range from two months to 30 years(N=112, one patient's history could not be verified). Prior totreatment, the mean years of heroin addiction was 10.1 years, with arange from one to 30 years (N=109, four patients' histories could not beverified). The mean daily methadone dose of opiate dependent patients instable treatment was 84 mg/day, with a range from 30 to 120 mg/day(N=106). Only patients with established stable doses were included inthis calculation; i.e., not on induction, increasing, tapering, orelimination schedules.

By sequencing PCR-amplified DNA from the study subjects, it wasdetermined that the previously reported sequence for the human mu opioidreceptor (8,9) was the most common allele found in the study population.Five different SNPs were also identified. For the purpose of this study,the term “most common” was used to denote the predominant mu opioidreceptor allele and the corresponding receptor that was originallyreported by cDNA cloning (8,9), and the term “variant” to denote theallelic genes/receptors containing polymorphic variations. Table 1 showsthese SNPs with information on the position of amino acid substitutionsand overall frequency of the variant alleles in the study population.Genotype and allele frequencies for the two most common allelicvariants, the A118G and C17T polymorphisms, are shown in Table 2. Theassociations of each frequency are broken down by ethnicity, gender, andopioid dependence. Since the number of individuals homozygous for theless common alleles was small, allele frequencies rather than genotypefrequencies were used to test for significant differences. Differencesof allele frequencies were tested among the three most common ethnicgroups, African-American, Caucasian, and Hispanic, irrespective ofopioid dependency Status. There was significant difference of allelefrequencies among ethnic groups for both the A118G [χ² ₍₂₎=7.15p=0.028)] and the C17T [χ² ₍₂₎=26.0 (p=0.000002)]. If the individualswho reported one parent from one ethnic group and one from anotherethnic group were excluded from this analysis, similar significancelevels were obtained for differences of both SNPs among ethnic groups.This result is not surprising since allele frequencies are known to varyamong ethnic groups. It is important to consider these differences whichcan confound association analyses. No significant association of genderwith either polymorphism was observed. For the A118G polymorphism, therewas no significant difference in allele frequencies between opioiddependent and non-dependent study subjects. However, the variant Tallele at the 17 position was present in a higher proportion of opiatedependent persons in the sample at a marginal significance level [Yatescorrected chi-square χ² ₍₁₎=3.70 (p=0.054).

This result is similar to that obtained in a previous study whichidentified this one SNP, and examined its frequency in association withdrug dependence (10). Table 3 shows the data stratified by ethnic groupand opiate dependency status for each of the A118G and C17Tpolymorphisms. The pooled Relative Risk (RR) and the Mantel-Haenszelchi-square (16) were calculated. For the A118G polymorphism there was nosignificant difference in allele frequencies between opioid dependentpersons and those with no history of drug abuse or dependence [RR=0.48χ² ₍₁₎=2.76 (p=0.096)]. Although not significant there was evidence ofheterogeneity between ethnic groups [RR=0.48 χ² ₍2)=5.16 (p=0.076)]. Itshould be noted that the direction of the Relative Risk less than onedenotes here that the A118G polymorphism was more frequent in normalhealthy volunteers with no history of drug dependence (controls) thanopioid dependent subjects (cases), and if present, the A118Gpolymorphism might confer some level of protection against opioiddependence, which is of particular interest given the functionaldifferences (see below). There was a marginally significant differencein the allele frequencies for the C17T polymorphism between cases andcontrols [RR=7.83 χ² ₍₁₎=3.73 (p=0.05)]. The test for heterogeneityamong ethnic groups was not significant [χ² ₍2)=3.95 (p=0.14)].

Cases and controls were examined for Hardy Weiberg equilibrium (HWE) byeach ethnic group individually and for all ethnic groups combined. Allgroups analyzed were in HWE, except for the C17T polymorphism for theopioid dependent subjects with all ethnic groups combined which showedsignificant deviation from HWE (p=0.008). Although under no obligationto provide a reason for this observation, and not intending to be boundby any postulation to explain this observation, these results may be dueto the admixture introduced by combining the different ethnic groupsinto one sample.

Furthermore, from the first group of individuals from whom the mu opioidreceptor has been sequenced (n=175), the following list of individualswith more than one variant form of the receptor were found: (1) Threeindividuals were double heterozygous for the A118G and C17T variants.The DNA sequencing methodology used cannot determine whether both SNPsare on a single chromosome or the two SNPs are on different chromosomes.These individuals would therefore be predicted either to express bothprototype receptors and receptors that have both amino acidsubstitutions, or, conversely, two variant receptor types, one with theA6V substitution and the other with the N40D substitution. (2) Oneindividual was a double heterozygote for the A118G SNP and G779A SNP.This individual would therefore be predicted either to express bothprototype receptors and receptors that have both amino acidsubstitutions, or, conversely, two variant receptor types, one with theN40D substitution and the other with the R260H substitution. (3) Oneindividual was homozygous for the C17T SNP and heterozygous for theG924A SNP. This individual is predicted to express variant receptorseach of which has the A6V substitution. However the G924A SNP would beuseful for ascribing a haplotype in this individual for genetichaplotype analysis.

The most prevalent genetic polymorphism identified is the A118G SNP witha substitution at the nucleotide position 118 with respect to the firstbase of the initiator codon for methionine (FIG. 1). This allele wasobserved in 29 of the 152 subjects, with 26 subjects being heterozygousand 3 being homozygous for the variant allele. This gives an allelefrequency of 10.5% in the subject population that we have examined forthis study. Nucleotide no. 118 is the first base in codon no. 40 of thehuman mu opioid receptor, and the A118G variant predicts an Asn to Aspchange in amino acid residue no. 40 of the receptor (N40D). The Asnresidue at amino acid position no. 40 in the most common mu receptor isa putative site for N-glycosylation (9); thus, the A118G variant wouldresult in the loss of a putative N-glycosylation site. The position ofamino acid 40 is in the N terminal region of the mu opioid receptor (9).Based on sequence motif similarities with other G protein-coupledreceptors (17), the N terminal region of opioid receptors, includingthat of the mu opioid receptor, is predicted to be in the extracellularspace (18). To explore any potential effects of the A118G polymorphismon the mu opioid receptor, position 118 of the most common mu receptorcDNA was mutated by site-directed mutagenesis, and a cDNA clone for thehuman mu opioid receptor containing the A118G variant was generated.This way, both the most common and the A118G variant receptors could beexpressed in cells to determine their cellular activity and theirbinding affinities.

Radioligand binding assays were performed with cell lines stablytransfected with either the A118G variant or the most common mureceptor, to determine whether the A118G polymorphism changes thereceptor's ability to bind opioid ligands, especially endogenous opioidpeptides, since they are the physiological agonists for the mu opioidreceptor. The A118G variant and the most common mu receptors yieldedsimilar binding affinity values for most of the opioid ligands tested,including the small endogenous peptide agonists Met- and Leu-enkephalin,each with five amino acid residues; endomorphin-1 and -2, each with fourresidues; the mu-selective synthetic opioid peptide DAMGO, with fiveamino acid residues; the endogenous ligand for the kappa opioid receptordynorphin A (1-17); as well as the mu-preferring opioid alkaloidagonists morphine, fentanyl, methadone, and the opioid antagonistnaloxone (FIG. 2, and data not shown) These results suggest that theA118G polymorphism does not change the overall binding properties of themu opioid receptor. This is not unexpected, since the predicted aminoacid change as a result of the A118G SNP is a single residuesubstitution in the N terminal region in the extracellular space, and isunlikely to drastically affect the overall tertiary structure of thereceptor.

There was a noticeable change, however, for the A118G variant receptorbinding of human β-endorphin, a much larger endogenous opioid peptide,which has 31 amino acid residues and which activates the mu opioidreceptor. Whereas the other, smaller, endogenous opioid peptides andalkaloid agonists and antagonist displayed similar binding affinitiesfor both receptors, the A 18G variant receptor showed higher bindingaffinity for β-endorphin than the most common receptor (FIG. 2), withthe ratio of the Ki of the most common to A118G variant being 3.46±0.31(mean±SEM, n=3). These results indicate that while the A118Gpolymorphism did not alter the overall profile of ligand binding to thereceptor, it specifically influenced the β-endorphin binding andresulted in tighter binding.

An important cellular activity of the mu opioid receptor is inhibitionof neuronal excitability by receptor-mediated inhibition of pre-synapticcalcium channels and activation of postsynaptic potassium channels (19,20). The major effector potassium channels for the mu receptor, as wellas for many other G protein-coupled receptors, are the Gprotein-activated inwardly rectifying K⁺ (GIRK) channels (21,22), andco-expression studies have shown that the mu opioid receptor can readilyactivate GIRK channels via a G protein-mediated mechanism (9,23,24). Toexamine the effect of the A 18G polymorphism, Xenopus oocyte expressionwas used to compare the A118G variant receptor with the most common muopioid receptor. Agonist stimulation of the A118G variant receptoractivated a potassium current similar to that seen with the most commonmu opioid receptor (9,23). The EC₅₀ values for endomorphin-I are 4.6 nMfor the most common receptor and 4.9 nM for the A118G variant receptor(FIG. 3), indicating that endomorphin-1 activated both receptors withsimilar potency. The EC₅₀ values for β-endorphin, however, differedabout three fold between the A118G variant and the most common mu opioidreceptors (FIG. 3), consistent with the change in the binding affinity(FIG. 2). These data indicate that, as a result of the SNP in thereceptor gene, the A118G variant receptor may be functionally differentfrom the most common mu opioid receptor.

An endogenous opioid with wide distribution in both the CNS and theperiphery, β-endorphin has been postulated to play a role in diversebiological functions (25-27). As a neuropeptide, it can modulateneurotransmitter actions in the CNS to mediate antinociception. It isalso a mediator in the stress response, of potential importance for thepathophysiology of the addictive diseases (28-36). β-endorphin canregulate the secretion of both stress and reproductive hormones, therebyinfluencing a variety of physiological functions. The synthesis andprocessing of β-endorphin is, in turn, regulated by other factors,including certain neurotransmitters and hormones. Given the diverseroles of β-endorphin, it is particularly interesting that the A118Gpolymorphism may change both the binding affinity and functional potencyof β-endorphin. On the basis of approximately three fold difference inthe affinity and potency values (FIG. 2 and 3), it is possible that twoindividuals with different mu opioid receptors (most common vs. A118Gvariant) may show variation in β-endorphin sensitivity. This, in turn,could alter perception of pain. It also could alter the vulnerability todevelop opioid addiction following exposure to opiates as well asaddictions to other drugs that alter the opioid system (2,6,37).

Binding and functional studies on the C17T receptor have shown thatβ-endorphin bound the C17T variant receptor less tightly than theprototype receptor. The average ratio of the IC₅₀ values was 0.72 forthe variant:prototype receptor, indicating a modestly lower affinity forthis ligand at the C17T variant receptor. This is in contrast to thehigher affinity of β-endorphin at the A118G variant receptor describedabove. One of the important indices of cellular functioning of the muopioid receptor is the inhibition of adenylyl cyclase activity followingagonist binding to the receptor. A modification of a previouslydescribed method (Chen, Y., J. Liu, and L. Yu. 1996. Functional couplingof a mu opioid receptor to G proteins and adenylyl cyclase: modulationby chronic morphine treatment. Addiction Biol. 1:49-59) was used formeasuring inhibition of adenylyl cyclase to reduce the amount ofvariability in the measurements. In this method, cells are replated 7 to9 hours before the cyclase assay is conducted. This time is sufficientfor the forskolin-stimulated cAMP levels and the percent inhibition bymu agonists to recover. The improvement in the intra-assay variabilityis most likely due to the fact that the number of cells in each well isthe same, since there has been little time for the growth rates todiverge between wells. Using this modified method, adenylyl cyclaseinhibition between the C17T and prototype receptors are compared (FIG.6). We have found that the C17T receptor is less sensitive toβ-endorphin: the EC50 values differed by a factor of approximately 2.7.The receptor was also less sensitive to leu-enkephalin: the EC50 valuesdiffered by a factor of approximately 2.8.

METHODS Study Subjects and Procedures

Addictive disease patients, specifically long-term heroin addictscurrently in chronic methadone maintenance treatment, and normal controlsubjects with no history of any drug or alcohol abuse, were extensivelycharacterized with respect to drug abuse, the addictive diseases,psychological and psychiatric profiles, and medical and ethnic familybackgrounds. Unrelated study subjects who were former heroin addictswere referred from methadone treatment clinics in the greater New YorkCity area, primarily those associated with The Biology of AddictiveDiseases Laboratory located at The Rockefeller University. These clinicsare the Adolescent Development Program and Adult Clinic at the New YorkHospital-Cornell Medical Center. Previously heroin-addicted patientsadmitted to the study conformed to the federally regulated criteria foradmission to a methadone maintenance program, that is, one or more yearsof daily multiple-dose self-administration of heroin or other opiateswith the development of tolerance, dependence, and drug-seeking behavior(38). Current or prior abuse of other drugs was not used as an exclusioncriterion for this group as long as opioid abuse continued to be theprimary diagnosis.

Unrelated healthy volunteer subjects were recruited primarily throughposting of notices and newspaper advertisements or referral byphysicians or staff at the Rockefeller University Hospital. Individualswith continuing drug or alcohol abuse or prior extended periods ofregular abuse were excluded from this category. The exclusion criteriawere defined as follows: for current or continuing abuse, alcohol, atleast five (for men) or four (for women) instances of drinking tointoxication during the previous 30 days; opiates, cocaine,amphetamines, or other illicit drugs (excluding cannabis), any useduring the previous 30 days. Users of nicotine or caffeine were notexcluded, nor were individuals who had abused cannabis for up to 12 daysduring the previous 30 days. For prior abuse, subjects were excluded whohad abused illicit drugs, excluding cannabis, for at least three times aweek for a period of at least one month. All study subjects wererigorously screened to assure appropriate characterization of addictivediseases, status of treatment, and presence or absence of polydrug oralcohol abuse. Subjects entering the study were required to be competentto understand the study procedures and understand and sign theInstitutional Review Board approved informed consent. Patients withschizophrenia or other psychotic mental illnesses were excluded from thestudy by this criterion. The presence of serological markers forhepatitis B, C or HIV was not used as an exclusion criterion.

Both addictive disease patients and normal volunteers admitted to thestudy were assessed by a psychiatrist or research nurse with severalpsychiatric and psychological instruments as well as the AddictionSeverity Index (39). Study subjects were also administered a detailedpersonal and medical and special addictive disease questionnaire as wellas a family history medical and addictive disease questionnaire designedto provide information regarding substance abuse and major mentalillness of first and second degree relatives. Study subjects provideddetailed information regarding family origin and ethnic background,including country or geographic area of birth. This information wasobtained for both the study subjects themselves and their immediateancestors (parents, grandparents and great-grandparents), to the extentthat the information was known by the study subjects. Study subjectswere classified into five groups: African-American, Caucasian, Hispanic(Caribbean and Central or South American origin), Native North American,and Other. The detailed ancestral information collected by the familyorigin questionnaire allowed classification of study subjects intodefined categories. Following psychiatric and behavioral assessment andinformed consent and family history acquisition, venipuncture on thestudy subject was performed, and a blood specimen was taken. Bloodsamples were processed for DNA extraction and EBV transformation tocreate stable cell lines that were stored for future studies. All bloodsamples were coded; the psychiatrists and nurses who performedpsychiatric and psychological assessments were blind to the genotypes ofthe study subjects, and the identity and categorization of the studysubjects was unknown to the laboratory research personnel.

Exon Amplification and Sequencing

Sequences for the non-coding regions of the human mu opioid receptorgene were used to design PCR primers. PCR primers were synthesized forthree of the four exons of the gene; the fourth exon was not included inthis study because this exon is small (4 or 12 amino acid residues) andalternative splicing m this exon has been shown to occur (40). Exon 1forward primer sequences were based on the 5′-untranslated region of thereceptor (9). Exon 1 reverse, exon 2 forward and reverse, and exon 3forward primer sequences were based on partial intron sequence dataobtained from inverse PCR of genomic DNA sequences for the receptor gene(data not shown). Exon 3 reverse primers were based on reported intron 3sequence (40). Two sets of primers were designed for each exon to allowfor nested PCR reactions to increase amplification specificity. Only onereverse primer was used for exon 1. The PCR reactions were performedwith 300-400 ng of genomic DNA, PCR products were separated on agarosegels, and the DNA fragments were purified for DNA sequencing DNApolymorphisms were confirmed by both manual and automated sequencing.

Mutagenesis

In vitro site-directed mutagenesis was performed to generate human muopioid receptor (hMOR) cDNA containing the A118G SNP. Complementaryoligonucleotides containing the desired mutation were synthesized andannealed to the pcDNA3 plasmid containing the most common allelic formof HMOR. Primer 1: TTGTCCCACTTAGATGGCGACCTGTCCGACCCA (SEQ ID NO:6).Primer 2: ACCGCATGGGTCGGACAGGTCGCCATCTAAGTG (SEQ ID NO:7). Primers wereextended and the product amplified by PCR using HMOR dsDNA as thetemplate, and Dpn I restriction enzyme was added afterwards to digestthe methylated, nonmutated most common dsDNA. After transformation intoE. coli cells, DNA from individual colonies was examined by restrictionenzyme digestion and DNA sequencing to confirm success of mutagenesis.

Cell Transfection and Binding Analysis

Stable transfection of the A118G SNP plasmid into AV-12 cells wasperformed as described (41). Individual colonies were then picked,expanded, and tested for expression levels by performing binding assays.Cells were harvested by washing with phosphate-buffered saline (PBS) atroom temperature, then they were scraped into homogenization solution(0.3 M sucrose, 25 mM Tris-HCl, pH 7.4, 0.05% BSA, and proteaseinhibitor cocktail, including 0.5 mM PMSF, 0.1 μg/ml leupeptin, and0.01% aprotinin), transferred to Dounce homogenizer and homogenized onice. The suspension was centrifuged at 1,000 g for 10 min and thesupernatant saved in a clean tube. The cell pellet was resuspended inhomogenization buffer, homogenized and centrifuged as described above.The supernatants from both extractions were combined and centrifuged at30,000 g for 20 min. The pelleted membranes were resuspended in bindingbuffer (50 mM Tris-HCl, pH 7.4), and binding assays were carried outusing membrane protein preparations as described (9).

Electrophysiology

Preparation of Xenopus oocytes was as previously reported (19). Oocyteswere injected with in vitro transcribed mRNAs for the most common orA118G variant mu opioid receptors together with the G protein-activatedinwardly rectifying K⁺ channels (GIRK1 and GIRK2). Two to three daysafter RNA injection, oocytes were voltage-clamped in ND96 solution (96mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.6) using atwo-electrode voltage-clamp (Axon Instruments). Cells were thensuperfused with a high potassium solution (98 mM KCl, 1 mM MgCl₂, 1.8 mMCaCl₂, 5 MM HEPES, pH 7.6), and stimulated with opioid ligands tomeasure the resulting potassium current.

References

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2. Kreek, M. J. Opiates, opioids and addiction. Mol. Psychiatry 1,232-254 (1996).

3. Pasternak, G. W. Pharmacological Mechanisms of opioid analgesics.Clin. Neuropharmacol. 16, 1-18 (1993).

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5. Zadina, J. E., Hackler, L., Ge, L. J., & Kastin, A. J. A potent andselective endogenous agonist for the mu-opiate receptor. Nature 386,499-502 (1997).

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33. Kosten, T. R., Kreek, M. J., Ragunath, J. & Kleber, H. D. (1986)Life Sci. 39, 55-59.

34. Kennedy, J. A., Hartman, N., Sbriglio, R., Khuri, E. & Kreek, M. J.(1990) Br. J. Addiction 85, 1133-1140.

35. Culpepper-Morgan, J. A., Twist, D. J., Petrillo, C. R., Soda, K. M.& Kreek, M. J. (1992) Metabolism: Clinical & Experimental 41, 578-581.

36. Culpepper-Morgan, J. A. & Kreek, M. J. (1997) Metabolism: Clinical &Experimental 46, 130-134.

37. Kreek, M. J. (1997) Pharmacol. Biochem. Behav. 57, 551-569.

39. McLellan, A. T., Luborsky, L., Woody, G. E. & O′Brien, C. P. (1980)J. Nerv. Ment. Dis. 168, 2633.

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The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

7 1 2162 DNA Homo sapiens misc_feature (2063) No feature for thisposition in GeneBank. 1 ggaattccgg ctataggcag aggagaatgt cagatgctcagctcggtccc ctccgcctga 60 cgctcctctc tgtctcagcc aggactggtt tctgtaagaaacagcaggag ctgtggcagc 120 ggcgaaagga agcggctgag gcgcttggaa cccgaaaagtctcggtgctc ctggctacct 180 cgcacagcgg tgcccgcccg gccgtcagta ccatggacagcagcgctgcc cccacgaacg 240 ccagcaattg cactgatgcc ttggcgtact caagttgctccccagcaccc agccccggtt 300 cctgggtcaa cttgtcccac ttagatggca acctgtccgacccatgcggt ccgaaccgca 360 ccaacctggg cgggagagac agcctgtgcc ctccgaccggcagtccctcc atgatcacgg 420 ccatcacgat catggccctc tactccatcg tgtgcgtggtggggctcttc ggaaacttcc 480 tggtcatgta tgtgattgtc agatacacca agatgaagactgccaccaac atctacattt 540 tcaaccttgc tctggcagat gccttagcca ccagtaccctgcccttccag agtgtgaatt 600 acctaatggg aacatggcca tttggaacca tcctttgcaagatagtgatc tccatagatt 660 actataacat gttcaccagc atattcaccc tctgcaccatgagtgttgat cgatacattg 720 cagtctgcca ccctgtcaag gccttagatt tccgtactccccgaaatgcc aaaattatca 780 atgtctgcaa ctggatcctc tcttcagcca ttggtcttcctgtaatgttc atggctacaa 840 caaaatacag gcaaggttcc atagattgta cactaacattctctcatcca acctggtact 900 gggaaaacct cgtgaagatc tgtgttttca tcttcgccttcattatgcca gtgctcatca 960 ttaccgtgtg ctatggactg atgatcttgc gcctcaagagtgtccgcatg ctctctggct 1020 ccaaagaaaa ggacaggaat cttcgaagga tcaccaggatggtgctggtg gtggtggctg 1080 tgttcatcgt ctgctggact cccattcaca tttacgtcatcattaaagcc ttggttacaa 1140 tcccagaaac tacgttccag actgtttctt ggcacttctgcattgctcta ggttacacaa 1200 acagctgcct caacccagtc ctttatgcat ttctggatgaaaacttcaaa cgatgcttca 1260 gagagttctg tatcccaacc tcttccaaca ttgagcaacaaaactccact cgaattcgtc 1320 agaacactag agaccacccc tccacggcca atacagtggatagaactaat catcagctag 1380 aaaatctgga agcagaaact gctccgttgc cctaacagggtctcatgcca ttccgacctt 1440 caccaagctt agaagccacc atgtatgtgg aagcaggttgcttcaagaat gtgtaggagg 1500 ctctaattct ctaggaaagt gcctactttt aggtcatccaacctctttcc tctctggcca 1560 ctctgctctg cacattagag ggacagccaa aagtaagtggagcatttgga aggaaaggaa 1620 tataccacac cgaggagtcc agtttgtgca agacacccagtggaaccaaa acccatcgtg 1680 gtatgtgaat tgaagtcatc ataaaaggtg acccttctgtctgtaagatt ttattttcaa 1740 gcaaatattt atgacctcaa caaagaagaa ccatcttttgttaagttcac cgtagtaaca 1800 cataaagtaa atgctacctc tgatcaaagc accttgaatggaaggtccga gtctttttag 1860 tgtttttgca agggaatgaa tccattattc tattttagacttttaacttc aacttaaaat 1920 tagcatctgg ctaaggcatc attttcacct ccatttcttggttttgtatt gtttaaaaaa 1980 aataacatct ctttcatcta gctccataat tgcaagggaagagattagca tgaaaggtaa 2040 tctgaaacac agtcatgtgt canctgtaga aaggttgattctcatgcact ncaaatactt 2100 ccaaagagtc atcatggggg atttttcatt cttaggctttcagtggtttg ttcctggaat 2160 tc 2162 2 400 PRT Homo sapiens 2 Met Asp SerSer Ala Ala Pro Thr Asn Ala Ser Asn Cys Thr Asp Ala 1 5 10 15 Leu AlaTyr Ser Ser Cys Ser Pro Ala Pro Ser Pro Gly Ser Trp Val 20 25 30 Asn LeuSer His Leu Asp Gly Asn Leu Ser Asp Pro Cys Gly Pro Asn 35 40 45 Arg ThrAsn Leu Gly Gly Arg Asp Ser Leu Cys Pro Pro Thr Gly Ser 50 55 60 Pro SerMet Ile Thr Ala Ile Thr Ile Met Ala Leu Tyr Ser Ile Val 65 70 75 80 CysVal Val Gly Leu Phe Gly Asn Phe Leu Val Met Tyr Val Ile Val 85 90 95 ArgTyr Thr Lys Met Lys Thr Ala Thr Asn Ile Tyr Ile Phe Asn Leu 100 105 110Ala Leu Ala Asp Ala Leu Ala Thr Ser Thr Leu Pro Phe Gln Ser Val 115 120125 Asn Tyr Leu Met Gly Thr Trp Pro Phe Gly Thr Ile Leu Cys Lys Ile 130135 140 Val Ile Ser Ile Asp Tyr Tyr Asn Met Phe Thr Ser Ile Phe Thr Leu145 150 155 160 Cys Thr Met Ser Val Asp Arg Tyr Ile Ala Val Cys His ProVal Lys 165 170 175 Ala Leu Asp Phe Arg Thr Pro Arg Asn Ala Lys Ile IleAsn Val Cys 180 185 190 Asn Trp Ile Leu Ser Ser Ala Ile Gly Leu Pro ValMet Phe Met Ala 195 200 205 Thr Thr Lys Tyr Arg Gln Gly Ser Ile Asp CysThr Leu Thr Phe Ser 210 215 220 His Pro Thr Trp Tyr Trp Glu Asn Leu ValLys Ile Cys Val Phe Ile 225 230 235 240 Phe Ala Phe Ile Met Pro Val LeuIle Ile Thr Val Cys Tyr Gly Leu 245 250 255 Met Ile Leu Arg Leu Lys SerVal Arg Met Leu Ser Gly Ser Lys Glu 260 265 270 Lys Asp Arg Asn Leu ArgArg Ile Thr Arg Met Val Leu Val Val Val 275 280 285 Ala Val Phe Ile ValCys Trp Thr Pro Ile His Ile Tyr Val Ile Ile 290 295 300 Lys Ala Leu ValThr Ile Pro Glu Thr Thr Phe Gln Thr Val Ser Trp 305 310 315 320 His PheCys Ile Ala Leu Gly Tyr Thr Asn Ser Cys Leu Asn Pro Val 325 330 335 LeuTyr Ala Phe Leu Asp Glu Asn Phe Lys Arg Cys Phe Arg Glu Phe 340 345 350Cys Ile Pro Thr Ser Ser Asn Ile Glu Gln Gln Asn Ser Thr Arg Ile 355 360365 Arg Gln Asn Thr Arg Asp His Pro Ser Thr Ala Asn Thr Val Asp Arg 370375 380 Thr Asn His Gln Leu Glu Asn Leu Glu Ala Glu Thr Ala Pro Leu Pro385 390 395 400 3 398 PRT Rattus norvegicus 3 Met Asp Ser Ser Thr GlyPro Gly Asn Thr Ser Asp Cys Ser Asp Pro 1 5 10 15 Leu Ala Gln Ala SerCys Ser Pro Ala Pro Gly Ser Trp Leu Asn Leu 20 25 30 Ser His Val Asp GlyAsn Gln Ser Asp Pro Cys Gly Leu Asn Arg Thr 35 40 45 Gly Leu Gly Gly AsnAsp Ser Leu Cys Pro Gln Thr Gly Ser Pro Ser 50 55 60 Met Val Thr Ala IleThr Ile Met Ala Leu Tyr Ser Ile Val Cys Val 65 70 75 80 Val Gly Leu PheGly Asn Phe Leu Val Met Tyr Val Ile Val Arg Tyr 85 90 95 Thr Lys Met LysThr Ala Thr Asn Ile Tyr Ile Phe Asn Leu Ala Leu 100 105 110 Ala Asp AlaLeu Ala Thr Ser Thr Leu Pro Phe Gln Ser Val Asn Tyr 115 120 125 Leu MetGly Thr Trp Pro Phe Gly Thr Ile Leu Cys Lys Ile Val Ile 130 135 140 SerIle Asp Tyr Tyr Asn Met Phe Thr Ser Ile Phe Thr Leu Cys Thr 145 150 155160 Met Ser Val Asp Arg Tyr Ile Ala Val Cys His Pro Val Lys Ala Leu 165170 175 Asp Phe Arg Thr Pro Arg Asn Ala Lys Ile Val Asn Val Cys Asn Trp180 185 190 Ile Leu Ser Ser Ala Ile Gly Leu Pro Val Met Phe Met Ala ThrThr 195 200 205 Lys Tyr Arg Gln Gly Ser Ile Asp Cys Thr Leu Thr Phe SerHis Pro 210 215 220 Thr Trp Tyr Trp Glu Asn Leu Leu Lys Ile Cys Val PheIle Phe Ala 225 230 235 240 Phe Ile Met Pro Val Leu Ile Ile Thr Val CysTyr Gly Leu Met Ile 245 250 255 Leu Arg Leu Lys Ser Val Arg Met Leu SerGly Ser Lys Glu Lys Asp 260 265 270 Arg Asn Leu Arg Arg Ile Thr Arg MetVal Leu Val Val Val Ala Val 275 280 285 Phe Ile Val Cys Trp Thr Pro IleHis Ile Tyr Val Ile Ile Lys Ala 290 295 300 Leu Ile Thr Ile Pro Glu ThrThr Phe Gln Thr Val Ser Trp His Phe 305 310 315 320 Cys Ile Ala Leu GlyTyr Thr Asn Ser Cys Leu Asn Pro Val Leu Tyr 325 330 335 Ala Phe Leu AspGlu Asn Phe Lys Arg Cys Phe Arg Glu Phe Cys Ile 340 345 350 Pro Thr SerSer Thr Ile Glu Gln Gln Asn Ser Thr Arg Val Arg Gln 355 360 365 Asn ThrArg Glu His Pro Ser Thr Ala Asn Thr Val Asp Arg Thr Asn 370 375 380 HisGln Leu Glu Asn Leu Glu Ala Glu Thr Ala Pro Leu Pro 385 390 395 4 372PRT Rattus norvegicus 4 Met Glu Pro Val Pro Ser Ala Arg Ala Glu Leu GlnPhe Ser Leu Leu 1 5 10 15 Ala Asn Val Ser Asp Thr Phe Pro Ser Ala PhePro Ser Ala Ser Ala 20 25 30 Asn Ala Ser Gly Ser Pro Gly Ala Arg Ser AlaSer Ser Leu Ala Leu 35 40 45 Ala Ile Ala Ile Thr Ala Leu Tyr Ser Ala ValCys Ala Val Gly Leu 50 55 60 Leu Gly Asn Val Leu Val Met Phe Gly Ile ValArg Tyr Thr Lys Leu 65 70 75 80 Lys Thr Ala Thr Asn Ile Tyr Ile Phe AsnLeu Ala Leu Ala Asp Ala 85 90 95 Leu Ala Thr Ser Thr Leu Pro Phe Gln SerAla Lys Tyr Leu Met Glu 100 105 110 Thr Trp Pro Phe Gly Glu Leu Leu CysLys Ala Val Leu Ser Ile Asp 115 120 125 Tyr Tyr Asn Met Phe Thr Ser IlePhe Thr Leu Thr Met Met Ser Val 130 135 140 Asp Arg Tyr Ile Ala Val CysHis Pro Val Lys Ala Leu Asp Phe Arg 145 150 155 160 Thr Pro Ala Lys AlaLys Leu Ile Asn Ile Cys Ile Trp Val Leu Ala 165 170 175 Ser Gly Val GlyVal Pro Ile Met Val Met Ala Val Thr Gln Pro Arg 180 185 190 Asp Gly AlaVal Val Cys Thr Leu Gln Phe Pro Ser Pro Ser Trp Tyr 195 200 205 Trp AspThr Val Thr Lys Ile Cys Val Phe Leu Phe Ala Phe Val Val 210 215 220 ProIle Leu Ile Ile Thr Val Cys Tyr Gly Leu Met Leu Leu Arg Leu 225 230 235240 Arg Ser Val Arg Leu Leu Ser Gly Ser Lys Glu Lys Asp Arg Ser Leu 245250 255 Arg Arg Ile Thr Arg Met Val Leu Val Val Val Gly Ala Phe Val Val260 265 270 Cys Trp Ala Pro Ile His Ile Phe Val Ile Val Trp Thr Leu ValAsp 275 280 285 Ile Asn Arg Arg Asp Pro Leu Val Val Ala Ala Leu His LeuCys Ile 290 295 300 Ala Leu Gly Tyr Ala Asn Ser Ser Leu Asn Pro Val LeuTyr Ala Phe 305 310 315 320 Leu Asp Glu Asn Phe Lys Arg Cys Phe Arg GlnLeu Cys Arg Ala Pro 325 330 335 Cys Gly Gly Gln Glu Pro Gly Ser Leu ArgArg Pro Arg Gln Ala Thr 340 345 350 Ala Arg Glu Arg Val Thr Ala Cys ThrPro Ser Asp Gly Pro Gly Gly 355 360 365 Gly Ala Ala Ala 370 5 380 PRTRattus norvegicus 5 Met Glu Ser Pro Ile Gln Ile Phe Arg Gly Glu Pro GlyPro Thr Cys 1 5 10 15 Ala Pro Ser Ala Cys Leu Leu Pro Asn Ser Ser SerTrp Phe Pro Asn 20 25 30 Trp Ala Glu Ser Asp Ser Asn Gly Ser Val Gly SerGlu Asp Gln Gln 35 40 45 Leu Glu Pro Ala His Ile Ser Pro Ala Ile Pro ValIle Ile Thr Ala 50 55 60 Val Tyr Ser Val Val Phe Val Val Gly Leu Val GlyAsn Ser Leu Val 65 70 75 80 Met Phe Val Ile Ile Arg Tyr Thr Lys Met LysThr Ala Thr Asn Ile 85 90 95 Tyr Ile Phe Asn Leu Ala Leu Ala Asp Ala LeuVal Thr Thr Thr Met 100 105 110 Pro Phe Gln Ser Ala Val Tyr Leu Met AsnSer Trp Pro Phe Gly Asp 115 120 125 Val Leu Cys Lys Ile Val Ile Ser IleAsp Tyr Tyr Asn Met Phe Thr 130 135 140 Ser Ile Phe Thr Leu Thr Met MetSer Val Asp Arg Tyr Ile Ala Val 145 150 155 160 Cys His Pro Val Lys AlaLeu Asp Phe Arg Thr Pro Leu Lys Ala Lys 165 170 175 Ile Ile Asn Ile CysIle Trp Leu Leu Ala Ser Ser Val Gly Ile Ser 180 185 190 Ala Ile Val LeuGly Gly Thr Lys Val Arg Glu Asp Val Asp Val Ile 195 200 205 Glu Cys SerLeu Gln Phe Pro Asp Asp Glu Tyr Ser Trp Trp Asp Leu 210 215 220 Phe MetLys Ile Cys Val Phe Val Phe Ala Phe Val Ile Pro Val Leu 225 230 235 240Ile Ile Ile Val Cys Tyr Thr Leu Met Ile Leu Arg Leu Lys Ser Val 245 250255 Arg Leu Leu Ser Gly Ser Arg Glu Lys Asp Arg Asn Leu Arg Arg Ile 260265 270 Thr Lys Leu Val Leu Val Val Val Ala Val Phe Ile Ile Cys Trp Thr275 280 285 Pro Ile His Ile Phe Ile Leu Val Glu Ala Leu Gly Ser Thr SerHis 290 295 300 Ser Thr Ala Val Leu Ser Ser Tyr Tyr Phe Cys Ile Ala LeuGly Tyr 305 310 315 320 Thr Asn Ser Ser Leu Asn Pro Val Leu Tyr Ala PheLeu Asp Glu Asn 325 330 335 Phe Lys Arg Cys Phe Arg Asp Phe Cys Phe ProIle Lys Met Arg Met 340 345 350 Glu Arg Gln Ser Thr Asn Arg Val Arg AsnThr Val Gln Asp Pro Ala 355 360 365 Ser Met Arg Asp Val Gly Gly Met AsnLys Pro Val 370 375 380 6 33 DNA Artificial Sequence Description ofArtificial Sequence Primer 6 ttgtcccact tagatggcga cctgtccgac cca 33 733 DNA Artificial Sequence Description of Artificial Sequence Primer 7accgcatggg tcggacaggt cgccatctaa gtg 33

What is claimed is:
 1. A method for determining a susceptibility in asubject to opioid at addiction comprising the steps of: a) removing abodily sample from said subject, wherein said sample comprises a firstand second allele comprising a human mu opioid receptor gene; b)determining whether said human mu opioid receptor gene of said firstallele comprises a DNA sequence having at least one variation in SEQ IDNO:1, wherein said variation comprises: A118G; or C17T, such that thepresence of said at least one variation in said human mu opioid receptorgene of said first allele is correlated with the subject'ssusceptibility to opioid addiction relative to the susceptibility toopioid addiction in a standard.
 2. The method for determining asusceptibility to opioid addiction in accordance with claim 1, furthercomprising the step of determining whether said human mu opioid receptorgene of said second allele comprises a DNA sequence having at least onevariation in SEQ ID NO:1, wherein said variation comprises: A 118G; orC17T, such that the presence of said at least one variation in saidhuman mu opioid receptor gene of said second allele is correlated withthe subject's susceptibility to opioid addiction relative to thesusceptibility to opioid addiction in said standard.
 3. The method ofeither of claims 1 or 2, wherein said at least one variation of saidhuman mu opioid receptor gene of said first and/or second allelecomprises A118G, and said variation correlated with a decreasedsusceptibility to opioid addiction relative to the susceptibility toopioid addiction in the standard.
 4. The method of either of claims 1nor 2, wherein where said at least one variation of said human mu opioidreceptor gene of said first and/or second allele comprises C17T, andsaid variation is correlated with increased susceptibility to opioidaddiction in said subject relative to susceptibility to opioid addictionin said standard.
 5. A method for determining a susceptibility to opioidaddiction in a subject relative to susceptibility in a standard,comprising the steps of: a) removing a bodily sample from said subject,wherein said sample comprises a human mu opioid receptor; b) determiningwhether said human mu opioid receptor comprises an amino acid sequencehaving at least one variation in SEQ ID NO:2, wherein said variationcomprises: Asn40Asp; or Ala6Val, such that the presence of said at leastone variation is correlated with the susceptibility to opioid addictionin said subject relative to susceptibility to opioid addiction in saidstandard, wherein the human mu opioid receptor of said standardcomprises an amino acid sequence of SEQ ID NO:2.
 6. The method of claim5, wherein said at least one variation in SEQ ID NO:2 comprisesAsn40Asp, and is correlated with a decreased susceptibility to opioidaddiction in said subject relative to said susceptibility to opioidaddiction in said standard, wherein the human mu opioid receptor of saidstandard comprises an amino acid sequence of SEQ ID NO:2.
 7. The methodof claim 5, wherein said at least one variation in SEQ ID NO:2 comprisesAla6Val, and is correlated with an increased susceptibility to opioidaddiction in said subject relative to susceptibility to opioid addictionin said standard, wherein said human mu opioid receptor of said standardcomprises an amino acid sequence of SEQ ID NO:2.